Methods of making arrays and artificial receptors

ABSTRACT

The present invention relates to artificial receptors and arrays or microarrays of artificial receptors or candidate artificial receptors. Each member of the array includes a plurality of building block compounds, typically immobilized in a spot on a support. The present invention also includes the building blocks, combinations of building blocks, arrays of building blocks, and receptors constructed of these building blocks together with a support. The present invention also includes methods of making and using these arrays and receptors.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional PatentApplication Serial No. 60/360,980 filed Mar. 1, 2002; 60/362,600, filedMar. 8, 2002; 60/375,655, filed Apr. 26, 2002; and 60/400,605, filedAug. 2, 2002.

INTRODUCTION

[0002] The present invention relates to artificial receptors, to methodsand compositions for making them, and to methods using them. A receptorprovides a binding site for and binds a ligand. For example, at anelementary level, receptors are often visualized having a binding siterepresented as a lock or site into which a key or ligand fits. Thebinding site is lined with, for example, hydrophobic or functionalgroups that provide favorable interactions with the ligand.

[0003] The present invention provides compositions and methods fordeveloping molecules that provide favorable interactions with a selectedligand. The present compositions and methods generate a wide variety ofmolecular structures, one or more of which interacts favorably with theselected ligand. Heterogeneous and immobilized combinations of buildingblock molecules form the variety of molecular structures. For example,combinations of 2, 3, 4, or 5 distinct building block moleculesimmobilized near one another on a support provide molecular structuresthat serve as candidate and working artificial receptors. FIG. 1schematically illustrates an embodiment employing 4 distinct buildingblocks in a spot on a microarray to make a ligand binding site. ThisFigure illustrates a group of 4 building blocks at the corners of asquare forming a unit cell. A group of four building blocks can beenvisioned as the vertices on any quadrilateral. FIG. 1 illustrates thatspots or regions of building blocks can be envisioned as multiple unitcells, in this illustration square unit cells. Groups of unit cells offour building blocks in the shape of other quadrilaterals can also beformed on a support.

[0004] Each immobilized building block molecule can provide one or more“arms” extending from a “framework” and each can include groups thatinteract with a ligand or with portions of another immobilized buildingblock. FIG. 2 illustrates that combinations of four building blocks,each including a framework with two arms (called “recognitionelements”), provides a molecular configuration of building blocks thatform a site for binding a ligand. Such a site formed by building blockssuch as those exemplified below can bind a small molecule, such as adrug, metabolite, pollutant, or the like, and/or can bind a largerligand such as a macromolecule or microbe.

BACKGROUND

[0005] The preparation of artificial receptors that bind ligands likeproteins, peptides, carbohydrates, microbes, pollutants,pharmaceuticals, and the like with high sensitivity and specificity isan active area of research. None of the conventional approaches has beenparticularly successful; achieving only modest sensitivity andspecificity mainly due to low binding affinity.

[0006] Antibodies, enzymes, and natural receptors generally have bindingconstants in the 10⁸-10¹² range, which results in both nanomolarsensitivity and targeted specificity. By contrast, conventionalartificial receptors typically have binding constants of about 10³ to10⁵, with the predictable result of millimolar sensitivity and limitedspecificity.

[0007] Several conventional approaches are being pursued in attempts toachieve highly sensitive and specific artificial receptors. Theseapproaches include, for example, affinity isolation, molecularimprinting, and rational and/or combinatorial design and synthesis ofsynthetic or semi-synthetic receptors.

[0008] Such rational or combinatorial approaches have been limited bythe relatively small number of receptors which are evaluated and/or bytheir reliance on a design strategy which focuses on only one buildingblock, the homogeneous design strategy. Common combinatorial approachesform microarrays that include 10,000 or 100,000 distinct spots on astandard microscope slide. However, such conventional methods forcombinatorial synthesis provide a single molecule per spot. Employing asingle building block in each spot provides only a single possiblereceptor per spot. Synthesis of thousands of building blocks would berequired to make thousands of possible receptors.

[0009] Further, these conventional approaches are hampered by thecurrently limited understanding of the principals which lead toefficient binding and the large number of possible structures forreceptors, which makes such an approach problematic.

[0010] There remains a need for methods and materials for makingartificial receptors that combines the efficiency of targeted synthesis,the spatial resolution of microarrays, and the exponential power ofcombinatorial display.

SUMMARY

[0011] The present invention relates to artificial receptors, arrays ormicroarrays of artificial receptors or candidate artificial receptors,and methods of making them. Each member of the array includes aplurality of building block compounds, typically immobilized in a spoton a support. The present invention also includes the building blocks,combinations of building blocks, arrays of building blocks, andreceptors constructed of these building blocks together with a support.The present invention also includes methods of using these arrays andreceptors.

[0012] The present invention includes and employs combinations of small,selected groups of building blocks in a combinatorial microarray displayformat to provide candidate artificial receptors. In an embodiment, thepresent invention employs up to about 4 building blocks, to make acandidate artificial receptor. Combinations of these building blocks canbe positioned on a substrate in configurations suitable for bindingligands such as proteins, peptides, carbohydrates, pollutants,pharmaceuticals, chemical warfare agents, microbes, and the like.

[0013] The present artificial receptors can be prepared by methodsincluding both focused combinatorial synthesis and targeted screeningarrays. The present compositions and methods can combine the advantagesof receptor focused synthesis and high throughput evaluation to rapidlyidentify and produce practical, target specific artificial receptors.

[0014] In an embodiment, the present invention includes a method ofmaking a heterogeneous building block array. This method includesforming a plurality of spots on a solid support, the spots including aplurality of building blocks, and coupling a plurality of buildingblocks to the solid support in the spots.

[0015] In an embodiment, the present invention includes a method ofusing an artificial receptor. This method includes contacting aheterogeneous building block array with a test ligand, detecting bindingof a test ligand to one or more spots in the array, and selecting one ormore of the binding spots as the artificial receptor. The artificialreceptor can be a lead or working artificial receptor. The method canalso include testing a plurality of building block arrays.

[0016] In an embodiment, the present invention includes a compositionincluding a support with a portion of the support comprising a pluralityof building blocks. The building blocks are coupled to the support. Thecomposition can include or be an artificial receptor, a heterogeneousbuilding block array, or a composition including a surface and a regionon the surface.

[0017] In an embodiment, the present invention includes an artificialreceptor including a plurality of building blocks coupled to a support.

[0018] In an embodiment, the present invention includes a heterogeneousbuilding block array. This array includes a support and a plurality ofspots on the support. The spots include a plurality of building blocks.The building blocks are coupled to the support.

[0019] In an embodiment, the present invention includes a compositionincluding a surface and a region on the surface. This region includes aplurality of building blocks, the building blocks being coupled to thesupport. In an embodiment, the present invention includes a compositionof matter including a plurality of building blocks.

[0020] In an embodiment, the building blocks include framework, linker,first recognition element, and second recognition element or have aformula linker-framework-(first recognition element)(second recognitionelement). The framework can be an amino acid. The building block canhave the formula:

[0021] in which: X, Y, Z, R₂, R₃, RE₁, RE₂ and L are describedhereinbelow.

BRIEF DESCRIPTION OF THE FIGURES

[0022]FIG. 1 schematically illustrates two dimensional representationsof an embodiment of a receptor according to the present invention thatemploys 4 different building blocks to make a ligand binding site.

[0023]FIG. 2 schematically illustrates two and three dimensionalrepresentations of an embodiment of a molecular configuration of 4building blocks, each building block including a recognition element, aframework, and a linker coupled to a support (immobilization/anchor).

[0024]FIG. 3A schematically illustrates representative structures of thesupport floor and building blocks according to the present invention ona surface of a support.

[0025]FIG. 3B schematically illustrates a support coupled to a signalelement, a building block, and a modified floor element.

[0026]FIG. 4 schematically illustrates representative space filingstructures of a candidate artificial receptor according to the presentinvention including both an amine floor and a four building blockreceptor.

[0027]FIG. 5 schematically illustrates a glass support including pendantamine or amide structures.

[0028]FIG. 6 schematically illustrates identification of a leadartificial receptor from among candidate artificial receptors.

[0029]FIG. 7 schematically illustrates employing successive subsets ofthe available building blocks to develop a lead or working artificialreceptor.

[0030]FIG. 8 schematically illustrates positional isomers ofcombinations of 4 building blocks (A, B, C, and D) at vertices of aquadrilateral, and such isomers on a scaffold. The representations ofthe positional isomers on a scaffold include building blocks A, B, C,and D and a sphere representing a ligand of interest.

[0031]FIG. 9 schematically illustrates serine as a framework for abuilding block and reactions for derivatizing the building block to addrecognition elements.

[0032]FIG. 10 schematically illustrates configurations in whichrecognition element(s), linker(s), and a chiral element can be coupledto a serine framework.

[0033]FIG. 11 schematically illustrates embodiments of the presentbuilding blocks forming a candidate artificial receptor having a regionsuitable for binding a test ligand.

[0034]FIG. 12 schematically illustrates embodiments of the presentbuilding blocks forming a candidate artificial receptor with a largermolecular footprint.

[0035]FIG. 13 schematically illustrates embodiments of the presentbuilding blocks forming a candidate artificial receptor that is shown assuitable for binding a test ligand with a cavity.

[0036]FIG. 14 schematically illustrates embodiments of HRP (Formula H1),HRP derivatives (Formulas H2 and H3), and conjugates of test ligand andHRP (Formulas H4, H5, and H6) useful in the present methods. Formula H4represents a conjugate of HRP with a chloroaromatic compound designated34K. Formula H5 represents a conjugate of HRP with an ethylene thioureadesignated ETU. ETU includes both aryl and heterocyclic moieties.Formula H5 represents a conjugate of HRP with a polycyclic chlorodioxinderivative designated TCDD.

[0037]FIG. 15A illustrates a key for the bar charts of FIGS. 15B-20 and22. This key identifies the bars for amino-glass, for acetylatedamino-glass, for each of homogeneous immobilized building blocks TyrA2B2(22), TyrA4B4 (44), and TyrA6B6 (66), and for candidate artificialreceptors TyrA2B2 plus TyrA4B4 (22/44); TyrA2B2 plus TyrA6B6 (22/66);TyrA4B4 plus TyrA6B6 (44/66); and TyrA2B2, TyrA4B4, plus TyrA6B6(22/44/66). Each bar for an artificial receptor including two buildingblocks is illustrated as 2 adjacent vertical stripes or segments. Thebar for an artificial receptor including three building blocks isillustrated as 3 adjacent vertical stripes or segments.

[0038]FIG. 15B illustrates bar charts of the binding pattern comparisonfor native HRP, acetylated amino-HRP, and the TCDD derivative ofamino-HRP. This Figure illustrates binding of this test ligand and thesecontrol derivatives to amino-glass, to acetylated amino-glass, to eachof homogeneous immobilized building blocks TyrA2B2, TyrA4B4, andTyrA6B6, and to candidate artificial receptors TyrA2B2 plus TyrA4B4;TyrA2B2 plus TyrA6B6; TyrA4B4 plus TyrA6B6; and TyrA2B2, TyrA4B4, plusTyrA6B6. The abbreviation for the building block including a linker, atyrosine framework, and recognition elements AxBy is TyrAxBy.

[0039]FIG. 16 illustrates bar charts showing the reproducibility of thebinding pattern for amino-HRP to amino-glass, to acetylated amino-glass,to each of homogeneous immobilized building blocks TyrA2B2, TyrA4B4, andTyrA6B6, and to candidate artificial receptors TyrA2B2 plus TyrA4B4;TyrA2B2 plus TyrA6B6; TyrA4B4 plus TyrA6B6; and TyrA2B2, TyrA4B4, plusTyrA6B6.

[0040]FIG. 17 illustrates bar charts of the binding pattern comparisonfor native HRP (Formula H1), amino-HRP (Formula H2), acetylatedamino-HRP (Formula H3), the 34K derivative of amino-HRP (Formula H4),the TCDD derivative of amino-HRP (Formula H6), and the ETU derivative ofamino-HRP (Formula H5). This Figure illustrates binding of these testligand conjugates and these control derivatives to amino-glass, toacetylated amino-glass, to each of homogeneous immobilized buildingblocks TyrA2B2, TyrA4B4, and TyrA6B6, and to candidate artificialreceptors TyrA2B2 plus TyrA4B4; TyrA2B2 plus TyrA6B6; TyrA4B4 plusTyrA6B6; and TyrA2B2, TyrA4B4, plus TyrA6B6.

[0041]FIG. 18 illustrates bar charts of the binding pattern comparisonfor the ETU derivative of amino-HRP (Formula H2) using kinetic andthermodynamic protocols for determining binding. This Figure illustratesbinding of this test ligand conjugate to amino-glass, to acetylatedamino-glass, to each of homogeneous immobilized building blocks TyrA2B2,TyrA4B4, and TyrA6B6, and to candidate artificial receptors TyrA2B2 plusTyrA4B4; TyrA2B2 plus TyrA6B6; TyrA4B4 plus TyrA6B6; and TyrA2B2,TyrA4B4, plus TyrA6B6.

[0042]FIG. 19 illustrates bar charts of the binding pattern comparisonfor the ETU derivative of amino-HRP (Formula H5) using protocols similarto those used in the experiments of FIG. 18. In the present experiment,the tubes were incubated for 1, 2, 4, 10, and 24 hours of incubation.

[0043]FIG. 20 illustrates the 3 bar charts (based on data presented inFIG. 16) along with LogP data for the test ligand.

[0044]FIG. 21 illustrates graphs of OD data for 0.1 μg/ml HRP-testligand conjugate versus LogP for the test ligand conjugate. The uppergraph in FIG. 21 plots the values for the n=1, homogeneous buildingblocks. The lower graph plots the values for candidate receptors. Inthis Figure, and throughout this application, TyrAB building blocks canbe further abbreviated as just the number of A and B. For example,TyrA4B4 can be abbreviated [44]. Candidate receptors including aplurality of building blocks can be similarly abbreviated. For example,a candidate receptor including TyrA4B4 plus TyrA6B6 can be abbreviated[44/66].

[0045]FIG. 22 illustrates bar charts comparing data for the candidatereceptors with combinations of 2 and 3 building blocks binding theacetylated amino HRP control and the three 34K, TCDD and ETU test ligandconjugates.

[0046]FIG. 23 schematically illustrates binding of acetylated amino HRPto derivatized-glass, to homogeneous immobilized building blocks, and tocandidate receptors. The candidate receptors include 5 building blocksin combinations of 2, 3, and 4. Table 9 lists the order in which resultsappear in FIGS. 23 and 24 for the floor tubes, immobilized buildingblocks, candidate receptors with combinations of 2 building blocks,candidate receptors with combinations of 3 building blocks, andcandidate receptors with combinations of 4 building blocks.

[0047]FIG. 24 schematically illustrates binding of amino-HRP-34K testligand conjugate (Formula H4) to derivatized-glass, to homogeneousimmobilized building blocks, and to candidate receptors identified inTable 9. The candidate receptors include 5 building blocks incombinations of 2, 3, and 4.

DETAILED DESCRIPTION

[0048] Definitions

[0049] A combination of building blocks immobilized on, for example, asupport can be a candidate artificial receptor, a lead artificialreceptor, or a working artificial receptor. That is, a heterogeneousbuilding block spot on a slide or a plurality of building blocks coatedon a tube or well can be a candidate artificial receptor, a leadartificial receptor, or a working artificial receptor. A candidateartificial receptor can become a lead artificial receptor, which canbecome a working artificial receptor.

[0050] As used herein the phrase “candidate artificial receptor” refersto an immobilized combination of building blocks that can be tested todetermine whether or not a particular test ligand binds to thatcombination. In an embodiment, the candidate artificial receptor can bea heterogeneous building block spot on a slide or a plurality ofbuilding blocks coated on a tube or well.

[0051] As used herein the phrase “lead artificial receptor” refers to animmobilized combination of building blocks that binds a test ligand at apredetermined concentration of test ligand, for example at 10, 1, 0.1,or 0.01 μg/ml, or at 1, 0.1, or 0.01 ng/ml. In an embodiment, the leadartificial receptor can be a heterogeneous building block spot on aslide or a plurality of building blocks coated on a tube or well.

[0052] As used herein the phrase “working artificial receptor” refers toa combination of building blocks that binds a test ligand with aselectivity and/or sensitivity effective for categorizing or identifyingthe test ligand. That is, binding to that combination of building blocksdescribes the test ligand as belonging to a category of test ligands oras being a particular test ligand. A working artificial receptor can,typically, bind the ligand at a concentration of, for example, 100, 10,1, 0.1, 0.01, or 0.001 ng/ml. In an embodiment, the working artificialreceptor can be a heterogeneous building block spot on a slide or aplurality of building blocks coated on a tube, well, slide, or othersupport or on a scaffold.

[0053] As used herein the phrase “working artificial receptor complex”refers to a plurality of artificial receptors, each a combination ofbuilding blocks, that binds a test ligand with a pattern of selectivityand/or sensitivity effective for categorizing or identifying the testligand. That is, binding to the several receptors of the complexdescribes the test ligand as belonging to a category of test ligands oras being a particular test ligand. The individual receptors in thecomplex can each bind the ligand at different concentrations or withdifferent affinities. Typically, the individual receptors in the complexeach bind the ligand at concentrations of 100, 10, 1, 0.1, 0.01 or 0.001ng/ml. In an embodiment, the working artificial receptor complex can bea plurality of heterogeneous building block spots or regions on a slide;a plurality of wells, each coated with a different combination ofbuilding blocks; or a plurality of tubes, each coated with a differentcombination of building blocks.

[0054] As used herein, the term “building block” refers to a molecularcomponent of an artificial receptor including portions that can beenvisioned as or that include one or more linkers, one or moreframeworks, and one or more recognition elements. In an embodiment, thebuilding block includes a linker, a framework, and one or morerecognition elements. The building block interacts with the ligand.

[0055] As used herein, the term “linker” refers to a portion of orfunctional group on a building block that can be employed to or thatdoes couple the building block to a support, for example, through acovalent link or electrostatic interactions.

[0056] As used herein, the term “framework” refers to a portion of abuilding block including the linker or to which the linker is coupledand to which one or more recognition elements are coupled.

[0057] As used herein, the term “recognition element” refers to aportion of a building block coupled to the framework but not covalentlycoupled to the support. Although not limiting to the present invention,the recognition element typically provides or forms one or more groups,surfaces, or spaces for interacting with the ligand.

[0058] As used herein, the phrase “plurality of building blocks” refersto two or more building blocks of different structure in a mixture, in akit, or on a support or scaffold. Each building block has a particularstructure, and use of building blocks in the plural, or of a pluralityof building blocks, refers to more than one of these particularstructures. Building blocks or plurality of building blocks does notrefer to a plurality of molecules each having the same structure.

[0059] As used herein, the phrase “combination of building blocks”refers to a plurality of building blocks that together are in a spot,region, or a candidate, lead, or working artificial receptor. Acombination of building blocks can be a subset of a set of buildingblocks. For example, a combination of building blocks can be one of thepossible combinations of 2, 3, 4, 5, or 6 building blocks from a set ofN (e.g., N=10-200) building blocks.

[0060] As used herein, the phrases “homogenous immobilized buildingblock” and “homogenous immobilized building blocks” refer to a supportor spot having immobilized on or within it only a single building block.

[0061] As used herein, the phrase “activated building block” refers to abuilding block activated to make it ready to form a covalent bond to afunctional group, for example, on a support. A building block includinga carboxyl group can be converted to a building block including anactivated ester group, which is an activated building block. Anactivated building block including an activated ester group can react,for example, with an amine to form a covalent bond.

[0062] As used herein, the term “immobilized” used with respect tobuilding blocks coupled to a support refers to building blocks beingstably oriented on the support so that they do not migrate on thesupport. Building blocks can be immobilized by covalent coupling, byionic interactions, or by electrostatic interactions, such as ionpairing.

[0063] As used herein a “region” of a support, tube, well, or surfacerefers to a contiguous portion of the support, tube, well, or surface.Building blocks coupled to a region typically refers to building blocksin proximity to one another in that region.

[0064] As used herein, a “bulky” group on a molecule is larger than amoiety including 7 or 8 carbon atoms.

[0065] As used herein, a “small” group on a molecule is hydrogen,methyl, or another group smaller than a moiety including 4 carbon atoms.

[0066] As used herein, the term “lawn” refers to a layer, spot, orregion of functional groups on a support, typically, at a densitysufficient to place coupled building blocks in proximity to one another.

[0067] The term “alkyl” refers to saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkylsubstituted alkyl groups. In certain embodiments, a straight chain orbranched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g.,C₁-C₁₂ for straight chain, C₁-C₆ for branched chain). Likewise,cycloalkyls typically have from 3-10 carbon atoms in their ringstructure, and preferably have 5, 6 or 7 carbons in the ring structure.

[0068] The term “alkyl” as used herein refers to both “unsubstitutedalkyls” and “substituted alkyls”, the latter of which refers to alkylmoieties having substituents replacing a hydrogen on one or more carbonsof the hydrocarbon backbone. Such substituents can include, for example,a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an ester, aformyl, or a ketone), a thiocarbonyl (such as a thioester, athioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphonate,a phosphinate, an amino, an amido, an amidine, an imine, a cyano, anitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, asulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aryl alkyl, oran aromatic or heteroaromatic moiety. The moieties substituted on thehydrocarbon chain can themselves be substituted, if appropriate. Forexample, the substituents of a substituted alkyl can include substitutedand unsubstituted forms of the groups listed above.

[0069] The phrase “aryl alkyl”, as used herein, refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

[0070] The terms “alkenyl” and “alkynyl” refer to unsaturated aliphaticgroups analogous in length and optional substitution to the alkylsgroups described above, but that contain at least one double or triplebond respectively.

[0071] The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics”. The aromatic ring can be substituted at one or morering positions with such substituents such as those described above foralkyl groups. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings (the rings are “fused rings”) wherein at leastone of the rings is aromatic, e.g., the other cyclic ring(s) can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

[0072] The terms “heterocyclyl” or “heterocyclic group” refer to 3- to12-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocyclylgroups include, for example, thiophene, thianthrene, furan, pyran,isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole,pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring can be substituted at one or more positionswith such substituents such as those described for alkyl groups.

[0073] The term “heteroatom” as used herein means an atom of any elementother than carbon or hydrogen, such as nitrogen, oxygen, sulfur andphosphorous.

[0074] Methods of Making and Using an Artificial Receptor

[0075] Making the Artificial Receptors

[0076] The present invention relates to a method of making an artificialreceptor or a candidate artificial receptor. In an embodiment, thismethod includes preparing a spot or region on a support, the spot orregion including a plurality of building blocks immobilized on thesupport. The method can include forming a plurality of spots on a solidsupport, each spot including a plurality of building blocks, andcoupling a plurality of building blocks to the solid support in eachspot. In an embodiment, an array of such spots is referred to as aheterogeneous building block array.

[0077] The building blocks can be activated to react with a functionalgroup on the support. Coupling can occur spontaneously after forming thespot of the building block or activated building block. The method caninclude mixing a plurality of activated building blocks and employingthe mixture in forming the spot(s). Alternatively, the method caninclude spotting individual activated building blocks on the support.

[0078] Forming a spot on a support can be accomplished by methods andapparatus such as pin spotters (sometimes referred to as printers),which can, for example, spot 10,000 to more than 100,000 spots on amicroscope slide. Other spotters include piezoelectric spotters (similarto ink jets) and electromagnetic spotters that can also spot, forexample, 10,000 to more than 100,000 spots on a microscope slide.Conventional mixing valves or manifolds can be employed to mix theactivated building blocks before spotting. These valves or manifolds canbe under control of conventional microprocessor based controllers forselecting building blocks and amounts of reagents.

[0079] Such spotting yields a microarray of spots of heterogeneouscombinations of building blocks, each of which can be a candidateartificial receptor. Each spot in a microarray includes a statisticallysignificant number of each building block. For example, although notlimiting to the present invention, it is believed that each micro spotof a size sufficiently small that 100,000 fit on a microscope slide caninclude approximately 320 million clusters of 4 building blocks.

[0080] In an embodiment, the present method includes making a receptorsurface. Making a receptor surface can include forming a region on asolid support, the region including a plurality of building blocks, andcoupling the plurality of building blocks to the solid support in theregion. The method can include mixing a plurality of activated buildingblocks and employing the mixture in forming the region or regions.Alternatively, the method can include applying individual activatedbuilding blocks in a region on the support. Forming a region on asupport can be accomplished, for example, by soaking a portion of thesupport with the building block solution.

[0081] In an embodiment, a tube or well coated with a support matrix canbe filled with activated building block (e.g., a solution containingactivated building block), which couples to the support matrix. Forexample, the support can be a glass tube or well coated with a pluralityof building blocks. The surface of the glass tube or well can be coatedwith a coating to which the plurality of building blocks becomecovalently bound. The resulting coating including building blocks can bereferred to as including heterogeneous building blocks.

[0082] Preferably, the method produces a surface or coating with adensity of building blocks sufficient to provide interactions of morethan one building block with a ligand. That is, the building blocks canbe in proximity to one another. Proximity of different building blockscan be detected by determining different (preferably greater) binding ofa test ligand to a surface including a plurality of building blockscompared to a surface or surfaces including only one of the buildingblocks.

[0083] The method can apply or spot building blocks onto a support incombinations of 2, 3, 4, or more building blocks. For an embodimentemploying a bulky tube or well, a manageable set of building blockspreferably provides fewer than several hundred or several thousandcombinations of building blocks. For example, in this context, a set of4, 5, or 6 building blocks provides a manageable number of combinationsof 2, 3, or 4 building blocks. In an embodiment, the method can beemployed to produce a plurality of tubes each tube having immobilized onits surface a heterogeneous combination of building blocks.

[0084] In an embodiment, the present method can be employed to produce asolid support having on its surface a plurality of regions or spots,each region or spot including a plurality of building blocks. Forexample, the method can include spotting a glass slide with a pluralityof spots, each spot including a plurality of building blocks. Such aspot can be referred to as including heterogeneous building blocks.

[0085] Each spot can include a density of building blocks sufficient toprovide interactions of more than one building block with a ligand. Suchinteractions can be determined as described above for regions. Themethod typically includes spotting the building blocks so that each spotis separated from the others. A plurality of spots of building blocks isreferred to herein as an array of spots.

[0086] In an embodiment, the method spots building blocks incombinations of 2, 3, 4, or more. The method can form up to 100,000 ormore spots on a glass slide. Therefore, in this embodiment of themethod, a manageable set of building blocks can provide several millioncombinations of building blocks. For example, in this context, a set of81 building blocks provides a manageable number of (1.66 million)combinations of 4 building blocks. For convenience in limiting thenumber of slides employed in the method, in this embodiment a setincludes up to 200 building blocks, preferably 50-100, preferably about80 (e.g., 81) building blocks.

[0087] In an embodiment, the method includes forming an array ofheterogeneous spots made from combinations of a subset of the totalbuilding blocks and/or smaller groups of the building blocks in eachspot. That is, the method forms spots including only, for example, 2 or3 building blocks, rather than 4 or 5. For example, the method can formspots from combinations of a full set of building blocks (e.g. 81 of aset of 81) in groups of 2 and/or 3. For example, the method can formspots from combinations of a subset of the building blocks (e.g., 25 ofthe set of 811) in groups of 4 or 5. For example, the method can formspots from combinations of a subset of the building blocks (e.g., 25 ofthe set of 81) in groups of 2 or 3. The method can include formingadditional arrays incorporating building blocks, lead artificialreceptors, or structurally similar building blocks.

[0088] In an embodiment, the method includes forming an array includingone or more spots that function as controls for validating or evaluatingbinding to artificial receptors of the present invention. In anembodiment, the method includes forming one or more regions, tubes, orwells that function as controls for validating or evaluating binding toartificial receptors of the present invention. Such a control spot,region, tube, or well can include no building block, only a singlebuilding block, only functionalized lawn, or combinations thereof.

[0089] The method can couple building blocks to supports using knownmethods for activating compounds of the types employed as buildingblocks and for coupling them to supports. Covalent coupling can produceartificial receptors sufficiently durable to be used repeatedly over aperiod of months. The method can employ building blocks includingactivated esters and couple them to supports including amine functionalgroups. The method can include activating a carboxyl group on a buildingblock by derivatizing to form the activated ester. By way of furtherexample, the method can couple building blocks including aminefunctional groups to supports including carboxyl groups. Pairs offunctional groups that can be employed on building blocks and supportsaccording to the method include nucleophile/electrophile pairs, such asamine and carboxyl (or activated carboxyl), thiol and maleimide, alcoholand carboxyl (or activated carboxyl), mixtures thereof, and the like.

[0090] The support can include any functional group suitable for forminga covalent bond with a building block. The support or the building blockcan include a functional group such as alcohol, phenol, thiol, amine,carbonyl, or like group. The support or the building block can include acarboxyl, alcohol, phenol, thiol, amine, carbonyl, maleimide, or likegroup that can react with or be activated to react with the support orthe building block. The support can include one or more of these groups.A plurality of building blocks can include a plurality of these groups.

[0091] The support or the building block can include a good leavinggroup bonded to, for example, an alkyl or aryl group. The leaving groupbeing “good” enough to be displaced by the alcohol, phenol, thiol,amine, carbonyl, or like group on the support or the building block.Such a support or the building block can include a moiety represented bythe formula: R—X, in which X is a leaving group such as halogen (e.g.,—Cl, —Br, or —I), tosylate, mesylate, triflate, and R is alkyl,substituted alkyl, cycloalkyl, heterocyclic, substituted heterocyclic,aryl alkyl, aryl, heteroaryl, or heteroaryl alkyl. The support caninclude one or more of these groups. A plurality of building blocks caninclude a plurality of these groups.

[0092] The method can employ any of the variety of known supportsemployed in combinatorial or synthetic chemistry (e.g., a microscopeslide, a bead, a resin, a gel, or the like). Suitable supports includefunctionalized glass, such as a functionalized slide or tube, glassmicroscope slide, glass plate, glass coverslip, glass beads, microporousglass beads, microporous polymer beads (e.g. those sold under thetradename Stratospheres™), silica gel supports, and the like.

[0093] The support typically includes a support matrix of a compound ormixture of compounds having functional groups suitable for coupling to abuilding block. The support matrix can be, for example, a coating on amicroscope slide or functionalizing groups on a bead, gel, or resin.Known support matrices are commercially available and/or include linkerswith functional groups that are coupled beads, gels, or resins. Thesupport matrix functional groups can be pendant from the support ingroups of one (e.g., as a lawn of amines, a lawn of another functionalgroup, or a lawn of a mixture of functional groups) or in groups of, forexample, 2, 3, 4, 5, 6, or 7. The groups of a plurality of functionalgroups pendant from the support can be visualized as or can be scaffoldmolecules pendant from the support.

[0094] The surface of the support can be visualized as including a floorand the building blocks (FIGS. 3A, 3B, and 4). As illustrated in FIG.3A, addition of building blocks to an amine lawn can proceed throughreaction of the amines to form building block amides with some of theamines remaining on the floor of the support or candidate artificialreceptor. Thus, the floor can be considered a feature of the candidateartificial receptor. The floor or modified floor can interact with theligand as part of the artificial receptor. The nucleophilic orelectrophilic groups on the floor can be left unreacted in theartificial receptor, or they can be modified. The floor can be modifiedwith a small group that alters the recognition properties of the floor(FIG. 3B). The floor can be modified with a signal element that producesa detectable signal when a test ligand is bound to the receptor (FIG.3B). For example, the signal element can be a fluorescent molecule thatis quenched by binding to the artificial receptor. For example, thesignal element can be a molecule that fluoresces only when bindingoccurs. The floor can be modified with a plurality of floor modifiers.For example, the floor can be modified with both a signal element and asmall group that alters the recognition properties of the floor.

[0095] In an embodiment, the candidate artificial receptor can includebuilding blocks and unmodified amines of the floor. Such a candidateartificial receptor has an amine/ammonium floor. In an embodiment, thecandidate artificial receptor can include building blocks and modifiedamines of the floor. For example, the floor amines can be modified bythe simplest amide modification of the amines to form the acetamide(e.g., by reacting with acetic anhydride or acetyl chloride).Alternatively, the floor amines can be modified by reaction withsuccinic anhydride, benzoyl chloride, and the like.

[0096] A lawn or other coating of functional groups can be derivatizedwith a maximum density of building blocks by exposing the lawn toseveral equivalents of activated building blocks. Typically, 10 or moreequivalents is sufficient for an adequate density of building blocks onthe support to observe building-block-dependent binding of a ligand. Anamine modified glass surface can be functionalized with building blocks,for example, by reaction with activated carboxyl derivatives to form anamide link to the lawn.

[0097] For example, a building block linker carboxyl group can beactivated by reacting the building block with carbodiimide in thepresence of sulfo N-hydroxysuccinimide in aqueous dimethylformamide. Theactivated building block can be reacted directly with an amine on aglass support (hereinafter amino glass). FIG. 3A illustrates thatderivatization of only a portion of the amine groups on the support canbe effective for producing candidate artificial receptors. Although notlimiting to the present invention, it is believed that the amine load onthe glass is in excess of that required for candidate artificialreceptor preparation. Preparations of surfaces including combinations ofbuilding blocks can be accomplished by, for example, premixing ofactivated building blocks prior to addition to the amino tube or thesequential mixing of the coupling solutions in the tubes.

[0098] A commercially available glass support can be prepared forcoupling building blocks by adding a support matrix to the surface ofthe support. The support matrix provides functional groups for couplingto the building block. Suitable support matrices include silanatingagents. For example a glass tube (e.g., a 12×75 mm borosilicate glasstube from VWR) can be coated to form a lawn of amines by reaction of theglass with a silanating agent such as 3-aminopropyltriethoxysilane.Building blocks including an activated ester can be bound to thiscoating by reaction of the building block activated ester with the amineglass to form the amide bound building block. Starting with acommercially available slide, an amino functionalized slide fromCorning, building blocks including an activated ester can be spotted onand covalently bound to the slide in a micro array by this samereaction. Such derivatization is illustrated in FIG. 5.

[0099] Using the Artificial Receptors

[0100] The present invention includes a method of using artificialreceptors. The present invention includes a method of screeningcandidate artificial receptors to find lead artificial receptors thatbind a particular test ligand. Detecting test ligand bound to acandidate artificial receptor can be accomplished using known methodsfor detecting binding to arrays on a slide or to coated tubes or wells.Typically, the method employs test ligand labeled with a detectablelabel, such as a fluorophore or an enzyme that produces a detectableproduct. Alternatively, the method can employ an antibody (or otherbinding agent) specific for the test ligand and including a detectablelabel. One or more of the spots that are labeled by the test ligand orthat are more or most intensely labeled with the test ligand areselected as lead artificial receptors. The degree of labeling can beevaluated by evaluating the signal strength from the label. Typically,the amount of signal is directly proportional to the amount of label andbinding. The test ligand can be a pure compound, a mixture, or a “dirty”mixture containing a natural product or pollutant. Such dirty mixturescan be tissue homogenate, biological fluid, soil sample, water sample,or the like. FIG. 6 provides a schematic illustration of an embodimentof this process.

[0101] According to the present method, screening candidate artificialreceptors against a test ligand can yield one or more lead artificialreceptors. One or more lead artificial receptors can be a workingartificial receptor. That is, the one or more lead artificial receptorscan be useful for detecting the ligand of interest as is. The method canthen employ the one or more artificial receptors as a working artificialreceptor for monitoring or detecting the test ligand. Alternatively, theone or more lead artificial receptors can be employed in the method fordeveloping a working artificial receptor. For example, the one or morelead artificial receptors can provide structural or other informationuseful for designing or screening for an improved lead artificialreceptor or a working artificial receptor. Such designing or screeningcan include making and testing additional candidate artificial receptorsincluding combinations of a subset of building blocks, a different setof building blocks, or a different number of building blocks.

[0102] In certain embodiments, the method of the present invention canemploy a smaller number of spots formed by combinations of a subset ofthe total building blocks and/or smaller groups of the building blocks.For example, the present method can employ an array including the numberof spots formed by combinations of 81 building blocks in groups of 2and/or 3. Then a smaller number of building blocks indicated by testcompound binding, for example 36 building blocks, can be tested in amicroarray with spots including larger groups, for example 4, of thebuilding blocks. Each set of microarrays can employ a different supportmatrix, lawn, or functionalized lawn. Such methods are schematicallyillustrated in FIG. 7.

[0103] For example, FIG. 7 illustrates that a single slide with the3,240 combinations of 2 building blocks that can be produced from a setof 81 building blocks can be used to define a subset of the buildingblocks. This subset of, e.g., 25, building blocks (which can be derivedfrom a 5×5 matrix of the results employing combinations of 2 buildingblocks), can be used to produce an additional 2,300 combinations of 3building blocks and/or 12,650 combinations of 4 building blocks. Thesecombinations from the subset can be screened to define the optimumreceptor configuration. The method can also include using combinationsof building blocks in different ratios in spots.

[0104] On a macro scale, an artificial receptor presented spot or regionincluding a plurality of building blocks has the plurality of buildingblocks distributed randomly throughout the spot or region. On amolecular scale, the distribution may not be random and even. Forexample, any selected group of only 2-10 building blocks may include agreater number of a particular building block or a particulararrangement of building blocks with respect to one another. A spot orregion with a random distribution makes a useful artificial receptoraccording to the present invention. Particular assortments of buildingblocks found in a random distribution can also make useful artificialreceptors.

[0105] An artificial receptor can include a particular assortment of acombination of 2, 3, 4, or more building blocks. Such an assortment canbe visualized as occupying positions on the surface of a support. Acombination of 2, 3, 4, or more building blocks can have each of thedifferent building blocks in distinct positions relative to one another.For example, building block 1 can be adjacent to any of building blocks2, 3, or 4. This can be illustrated by considering the building blocksat the vertices of a polygon. For example, FIG. 8 illustrates positionalisomers of 4 different building blocks at the vertices of aquadrilateral.

[0106] In an embodiment of the method, a candidate artificial receptorcan be optimized to a preferred lead or working artificial receptor bymaking one or more of the positional isomers and determining its abilityto bind the test ligand of interest. Advantageously, the positionalisomers can be made on a scaffold (FIG. 8). Scaffold positional isomerartificial receptors can be made, for example, on a scaffold withmultiple functional groups that can be protected and deprotected byorthogonal chemistries. The scaffold positional isomer lead artificialreceptors can be evaluated by any of a variety of methods suitable forevaluating binding of ligands to scaffold receptors. For example, thescaffold lead artificial receptors can be chromatographed againstimmobilized test ligand.

[0107] In an embodiment, the method of using an artificial receptorincludes contacting a first heterogeneous molecular array with a testligand. The array can include a support and a plurality of spots ofbuilding blocks attached to the support. In the array, each spot ofbuilding blocks can include a plurality of building blocks with eachbuilding block being coupled to the support. The method includesdetecting binding of a test ligand to one or more spots; and selectingone or more of the binding spots as the artificial receptor.

[0108] In this embodiment, the building blocks in the array can define afirst set of building blocks, and the plurality of building blocks ineach binding spot defines one or more selected binding combinations ofbuilding blocks. The first set of building blocks can include or be asubset of a larger set of building blocks. In an embodiment, the spotsof building blocks can include 2, 3, or 4 building blocks. The first setcan be immobilized using a first support matrix, a first lawn, or afirst functionalized lawn.

[0109] In the method, the artificial receptor can include or be one ormore lead artificial receptors. In the method, the artificial receptorscan include or be one or more working artificial receptors.

[0110] This embodiment of the method can also include determining thecombinations of building blocks in the one or more binding spots. Thesecombinations can be used as the basis for developing one or moredeveloped combinations of building blocks distinct from those in the oneor more selected combinations of building blocks. This embodimentcontinues with contacting the test ligand with a second heterogeneousmolecular array comprising a plurality of spots, each spot comprising adeveloped combination of building blocks; detecting binding of a testligand to one or more spots of the second heterogeneous molecular array;and selecting one or more of the spots of the second heterogeneousmolecular array as the artificial receptor. The second set can beimmobilized using a second support matrix, a second lawn, or a secondfunctionalized lawn different from those used with the first set.

[0111] In this embodiment, the building blocks in the secondheterogeneous molecular array define a second set of building blocks.The first set of building blocks can include or be a subset of a largerset of building blocks and/or the second subset of building blocks caninclude or define a subset of the larger set of building blocks.Advantageously, the first subset is not equivalent to the second subset.In an embodiment, the spots of the second heterogeneous molecular arraycan include 3, 4, or 5 building blocks, and/or the spots of the secondheterogeneous molecular array can include more building blocks than thebinding spots.

[0112] The artificial receptor can include or be a lead artificialreceptor. The artificial receptor can include or be one or more workingartificial receptors. The method can also include varying the structureof the lead artificial receptor to increase binding speed or bindingaffinity of the test ligand.

[0113] In an embodiment, the method includes identifying the pluralityof building blocks making up the artificial receptor. The identifiedplurality of building blocks can then be coupled to a scaffold moleculeto make a scaffold artificial receptor. This scaffold artificialreceptor can be evaluated for binding of the test ligand. In anembodiment, coupling the identified plurality of building blocks to thescaffold can include making a plurality of positional isomers of thebuilding blocks on the scaffold. Evaluating the scaffold artificialreceptor can then include comparing the plurality of the scaffoldpositional isomer artificial receptors. In this embodiment, one or moreof the scaffold positional isomer artificial receptors can be selectedas one or more lead or working artificial receptors.

[0114] In an embodiment, the method includes screening a test ligandagainst an array including one or more spots that function as controlsfor validating or evaluating binding to artificial receptors of thepresent invention. In an embodiment, the method includes screening atest ligand against one or more regions, tubes, or wells that functionas controls for validating or evaluating binding to artificial receptorsof the present invention. Such a control spot, region, tube, or well caninclude no building block, only a single building block, onlyfunctionalized lawn, or combinations thereof.

[0115] Embodiments of Artificial Receptors

[0116] A candidate artificial receptor, a lead artificial receptor, or aworking artificial receptor includes combination of building blocksimmobilized on, for example, a support. An individual artificialreceptor can be a heterogeneous building block spot on a slide or aplurality of building blocks coated on a tube or well.

[0117] An array of candidate artificial receptors can be a commercialproduct sold to parties interested in using the candidate artificialreceptors as implements in developing receptors for test ligands ofinterest. In an embodiment, a useful array of candidate artificialreceptors includes a plurality of glass slides, the glass slidesincluding spots of all combinations of members of a set of buildingblocks, each combination including a predetermined number of buildingblocks. In an embodiment, a useful group of candidate artificialreceptors includes a plurality of tubes or wells, each with a coating ofa plurality of immobilized building blocks.

[0118] One or more lead artificial receptors can be developed from aplurality of candidate artificial receptors. In an embodiment, a leadartificial receptor includes a combination of building blocks and bindsdetectable quantities of test ligand upon exposure to, for example,several picomoles of test ligand at a concentration of 1, 0.1, or 0.01μg/ml, or at 1, 0.1, or 0.01 ng/ml test ligand; at a concentration of0.01 μg/ml, or at 1, 0.1, or 0.01 ng/ml test ligand; or a concentrationof 1, 0.1, or 0.01 ng/ml test ligand.

[0119] Artificial receptors, particularly candidate or lead artificialreceptors, can be in the form of an array of artificial receptors. Suchan array can include, for example, 1.66 million spots, each spotincluding one combination of 4 building blocks from a set of 81 buildingblocks. Each spot is a candidate artificial receptor and a combinationof building blocks. The array can also be constructed to include leadartificial receptors. For example, the array of artificial receptors caninclude combinations of fewer building blocks and/or a subset of thebuilding blocks.

[0120] In an embodiment, an array of candidate artificial receptorsincludes building blocks of general Formula 2 (shown hereinbelow), withRE₁ being B1, B2, B3, B4, B5, B6, B7, B8, or B9 (shown hereinbelow) andwith RE₂ being A1, A2, A3, A4, A5, A6, A7, A8, or A9 (shownhereinbelow). Preferably the framework is tyrosine.

[0121] One or more working artificial receptors can be developed fromone or more lead artificial receptors. In an embodiment, a workingartificial receptor includes a combination of building blocks and bindscategorizing or identifying quantities of test ligand upon exposure to,for example, several picomoles of test ligand at a concentration of 100,10, 1, 0.1, 0.01, or 0.001 ng/ml test ligand; at a concentration of 10,1, 0.1, 0.01, or 0.001 ng/ml test ligand; or a concentration of 1, 0.1,0.01, or 0.001 ng/ml test ligand.

[0122] In an embodiment, the artificial receptor of the inventionincludes a plurality of building blocks coupled to a support. In anembodiment, the plurality of building blocks can include or be buildingblocks of Formula 2 (shown below). In an embodiment, the plurality ofbuilding blocks can include or be building blocks of formula TyrA2B2and/or TyrA4B4 (shown below; the abbreviation for the building blockincluding a linker, a tyrosine framework, and recognition elements AxByis TyrAxBy). In an embodiment, the plurality of building blocks caninclude or be building blocks of formula TyrA4B2 and/or TyrA4B4 (shownbelow). In an embodiment, the plurality of building blocks can includeor be building blocks of formula TyrA2B2, TyrA4B2, TyrA4B4, and/orTyrA6B6 (shown below).

[0123] In an embodiment, a candidate artificial receptor can includecombinations of building blocks of formula TyrA2B2, TyrA4B4, or TyrA6B6.In an embodiment, a candidate artificial receptor can includecombinations of building blocks of formula TyrA2B2, TyrA4B4, TyrA6B6,TyrA4B2, or TyrA4B6. In an embodiment, a candidate artificial receptorcan include combinations of building blocks of formula TyrA2B2, TyrA2B4,TyrA4B2, TyrA4B4, TyrA4B6, TyrA6B4, TyrA6B6, TyrA6B8, TyrA8B6, orTyrA8B8.

[0124] Working Receptor Systems

[0125] In an embodiment, a working artificial receptor or workingartificial receptor complex can be incorporated into a system or devicefor detecting a ligand of interest. Binding of a ligand of interest to aworking artificial receptor or complex can produce a detectable signal,for example, through mechanisms and properties such as scattering,absorbing or emitting light, producing or quenching fluorescence orluminescence, producing or quenching an electrical signal, and the like.Spectroscopic detection methods include use of labels or enzymes toproduce light for detection by optical sensors or optical sensor arrays.The light can be ultraviolet, visible, or infrared light, which can beproduced and/or detected through fluorescence, fluorescencepolarization, chemiluminescence, bioluminescence, orchemibioluminescence. Systems and methods for detecting electricalconduction, and changes in electrical conduction, include ellipsometry,surface plasmon resonance, capacitance, conductometry, surface acousticwave, quartz crystal microbalance, love-wave, infrared evanescent wave,enzyme labels with electrochemical detection, nanowire field effecttransistors, MOSFETS—metal oxide semiconductor field effect transistors,CHEMFETS—organic membrane metal oxide semiconductor field effecttransistors, ICP intrinsically conducting polymers, FRET—fluorescenceresonance energy transfer.

[0126] Apparatus that can detect such binding to or signal from aworking artificial receptor or complex includes UV, visible or Infraredspectrometer, fluorescence or luminescence spectrometer, surface plasmonresonance, surface acoustic wave or quartz crystal microbalancedetectors, pH, voltammetry or amperometry meters, radioisotope detector,or the like.

[0127] In such an apparatus, a working artificial receptor or complexcan be positioned on a light fiber to provide a detectable signal, suchas an increase or decrease in transmitted light, reflected light,fluorescence, luminescence, or the like. The detectable signal canoriginate from, for example, a signaling moiety incorporated into theworking artificial receptor or complex or a signaling moiety added tothe working artificial receptor. The signal can also be intrinsic to theworking artificial receptor or to the ligand of interest. The signal cancome from, for example, the interaction of the ligand of interest withthe working artificial receptor, the interaction of the ligand ofinterest with a signaling moiety which has been incorporated into theworking artificial receptor, into the light fiber, onto the light fiber.

[0128] In an embodiment of the system, more than one working artificialreceptor, arranged as regions or spots in an array, is on the surface ofa support, such as a glass plate. The ligand or ligands of interest or asample suspected of containing the ligand or ligands of interest (e.g.,a sample containing a mixture of DNA segments or fragments, proteins orprotein fragments, carbohydrates or carbohydrate fragments, or the like)is brought into contact with the working artificial receptors or array.Contact can be achieved by addition of a solution of the ligand orligands of interest or a sample suspected of containing the ligand orligands of interest. A detectable fluorescence signal can be produced bya signaling moiety incorporated into the working artificial receptorarray or a signaling moiety which is added to the ligand or ligands ofinterest or the sample suspected of containing the ligand or ligands ofinterest. The fluorescent moieties produce a signal for each workingartificial receptor in the array, which produces a pattern of signalresponse which is characteristic of the composition of the sample ofinterest.

[0129] In an embodiment of the system, more than one working artificialreceptor, arranged as regions or spots in an array, is on a support,such as a glass or plastic surface. The surface can be incorporated ontothe signaling surfaces of one or more surface plasmon resonancedetectors. The ligands of interest or a sample suspected of containingthe ligands of interest (e.g., a sample containing a mixture of DNAsegments or fragments, proteins or protein fragments, carbohydrates orcarbohydrate fragments, or the like) is brought into contact with theworking artificial receptors or array. Contacting can be accomplished byaddition of a solution of the ligands of interest or a sample suspectedof containing the ligands of interest. Detectable electrical signals canbe produced by binding of the ligands of interest to the workingartificial receptors array on the surface of the surface plasmonresonance detectors. Such detectors produce a signal for each workingartificial receptor in the array, which produces a pattern of signalresponse, which is characteristic of the composition of the sample ofinterest.

[0130] In an embodiment of the system, the working artificial receptoris on a support such as the inner surface of a test tube, microwell,capillary, microchannel, or the like. The ligand of interest or a samplesuspected of containing the ligand of interest is brought into contactwith the working artificial receptor or complex by addition of asolution containing the ligand of interest or a sample suspected ofcontaining the ligand of interest. A detectable colorimetric,fluorometric, radiometric, or the like, signal is produced by acolorimetric, enzyme, fluorophore, radioisotope, metal ion, or the like,labeled compound or conjugate of the ligand of interest. This labeledmoiety can be reacted with the working artificial receptor or complex incompetition with the solution containing the ligand of interest or thesample suspected of containing the ligand of interest.

[0131] In an embodiment of the system, the working artificial receptoris on a support such as the surface of a surface acoustic wave or quartzcrystal microbalance or surface plasmon resonance detector. The ligandof interest or a sample suspected of containing the ligand of interestcan be brought into contact with the working artificial receptor orcomplex by exposure to a stream of air, to an aerosol, or to a solutioncontaining the ligand of interest or a sample suspected of containingthe ligand of interest. A detectable electrical signal can be producedby the interaction of the ligand of interest with the working artificialreceptor or complex on the active surface of the surface acoustic waveor quartz crystal microbalance or surface plasmon resonance detector.

[0132] In an embodiment of the system, the more than one workingartificial receptor, arranged as a series of discrete areas or spots orzones or the like, is on the surface of a light fiber. The ligand ofinterest or a sample suspected of containing the ligand of interest canbe brought into contact with the working artificial receptor or complexby exposure to a stream of air, to an aerosol, or to a solutioncontaining the ligand of interest or a sample suspected of containingthe ligand of interest. A detectable colorimetric, fluorometric, or likesignal can be produced by a label incorporated into the light fibersurface. The colorimetric or fluorogenic signal can be intrinsic to theligand, or can be an inherent colorimetric or fluorogenic signalproduced on binding of the ligand to the working artificial receptors.

[0133] An embodiment of the system, combines the artificial receptorswith nanotechnology derived nanodevices to give the devices the abilityto bind (“see”), bind and incorporate (“eat”), or modify (“use inmanufacture”) the target material. In an embodiment of the system, theworking artificial receptor is incorporated into or on a nanodevice. Theligand of interest or a sample suspected of containing the ligand ofinterest can be brought into contact with the working artificialreceptor nanodevice by addition of the nanodevice to an air or water orsoil or biological fluid or cell or biological tissue or biologicalorganism or the like. A detectable signal can be produced by a suitablesensor on the nanodevice and a desired action like a radio signal orchemical reaction or mechanical movement or the like is produced by thenanodevice in response to the ligand of interest.

[0134] The present artificial receptors can be part of products used in:analyzing a genome and/or proteome; pharmaceutical development;detectors for any of the test ligands; drug of abuse diagnostics ortherapy; hazardous waste analysis or remediation; chemical warfare alertor intervention; disease diagnostics or therapy; cancer diagnostics ortherapy; biowarfare alert or intervention; food chain contaminationanalysis or remediation; and the like.

[0135] More specifically, the present artificial receptors can be usedin products for identification of sequence specific small moleculeleads; protein isolation and identification; identification of proteinto protein interactions; detecting contaminants in food or foodproducts; clinical analysis of food contaminants; clinical analysis ofprostate specific antigen; clinical and field or clinical analysis ofcocaine; clinical and field or clinical analysis of other drugs ofabuse; other clinical analysis systems, home test systems, or fieldanalysis systems; monitors or alert systems for bioterrorism or chemicalwarfare agents; and the like.

[0136] Test Ligands

[0137] The test ligand can be any ligand for which binding to an arrayor surface can be detected. Test ligands include prostate specificantigen, other cancer markers, insulin, warfarin, other anti-coagulants,cocaine, other drugs-of-abuse, markers for E. coli, markers forSalmonella sp., markers for other food-borne toxins, food-borne toxins,markers for Smallpox virus, markers for anthrax, markers for otherpossible bioterrorism agents, pharmaceuticals and medicines, pollutantsand chemicals in hazardous waste, chemical warfare agents, markers ofdisease, pharmaceuticals, pollutants, biologically important cations(e.g., potassium or calcium ion), peptides, carbohydrates, enzymes,bacteria, viruses, and the like.

[0138] Building Blocks

[0139] The present invention relates to building blocks for making orforming candidate artificial receptors. Building blocks are designed,made, and selected to provide a variety of structural characteristicsamong a small number of compounds. A building block can provide one ormore structural characteristics such as positive charge, negativecharge, acid, base, electron acceptor, electron donor, hydrogen bonddonor, hydrogen bond acceptor, free electron pair, π electrons, chargepolarization, hydrophilicity, hydrophobicity, and the like. A buildingblock can be bulky or it can be small.

[0140] A building block can be visualized as including severalcomponents, such as one or more frameworks, one or more linkers, and/orone or more recognition elements. The framework can be covalentlycoupled to each of the other building block components. The linker canbe covalently coupled to the framework and to a support. The recognitionelement can be covalently coupled to the framework. In an embodiment, abuilding block includes a framework, a linker, and a recognitionelement. In an embodiment, a building block includes a framework, alinker, and two recognition elements. A building block including aframework, a linker, and one or more recognition elements can beschematically represented as:

[0141] Framework

[0142] The framework can be selected for functional groups that providefor coupling to the recognition moiety and for coupling to or being thelinking moiety. The framework can interact with the ligand as part ofthe artificial receptor. Typically, the framework includes multiplereaction sites with orthogonal and reliable functional groups and withcontrolled stereochemistry. Suitable functional groups with orthogonaland reliable chemistries include, for example, carboxyl, amine,hydroxyl, phenol, carbonyl, and thiol groups, which can be individuallyprotected, deprotected, and derivatized. Typically, the framework hastwo, three, or four functional groups with orthogonal and reliablechemistries.

[0143] A framework including three sites for orthogonal and reliablechemistries can be schematically represented as:

[0144] The three functional groups can be independently selected, forexample, from carboxyl, amine, hydroxyl, phenol, carbonyl, or thiolgroup. The framework can include alkyl, substituted alkyl, cycloalkyl,heterocyclic, substituted heterocyclic, aryl alkyl, aryl, heteroaryl,heteroaryl alkyl, and like moieties.

[0145] A general structure for a framework with three functional groupscan be represented by Formula 1a:

[0146] A general structure for a framework with four functional groupscan be represented by Formula 1b:

[0147] In these general structures: R₁ can be a 1-12, preferably 1-6,preferably 1-4 carbon alkyl, substituted alkyl, cycloalkyl,heterocyclic, substituted heterocyclic, aryl alkyl, aryl, heteroaryl,heteroaryl alkyl, or like group; and F₁, F₂, F₃, or F₄ can independentlybe a carboxyl, amine, hydroxyl, phenol, carbonyl, or thiol group. F₁,F₂, F₃, or F₄ can independently be a 1-12, preferably 1-6, preferably1-4 carbon alkyl, substituted alkyl, cycloalkyl, heterocyclic,substituted heterocyclic, aryl alkyl, aryl, heteroaryl, heteroarylalkyl, or inorganic group substituted with carboxyl, amine, hydroxyl,phenol, carbonyl, or thiol group. F₃ and/or F₄ can be absent.

[0148] A variety of compounds fit the schemes and formulas describingthe framework including amino acids, and naturally occurring orsynthetic compounds including, for example, oxygen and sulfur functionalgroups. The compounds can be racemic or optically active. For example,the compounds can be natural or synthetic amino acids, α-hydroxy acids,thioic acids, and the like.

[0149] Suitable molecules for use as a framework include a natural orsynthetic amino acid, particularly an amino acid with a functional group(e.g., third functional group) on its side chain. Amino acids includecarboxyl and amine functional groups. The side chain functional groupcan include, for natural amino acids, an amine (e.g., alkyl amine,heteroaryl amine), hydroxyl, phenol, carboxyl, thiol, thioether, oramidino group. Natural amino acids suitable for use as frameworksinclude, for example, serine, threonine, tyrosine, aspartic acid,glutamic acid, asparagine, glutamine, cysteine, lysine, arginine,histidine. Synthetic amino acids can include the naturally occurringside chain functional groups or synthetic side chain functional groupswhich modify or extend the natural amino acids with alkyl, substitutedalkyl, cycloalkyl, heterocyclic, substituted heterocyclic, aryl alkyl,aryl, heteroaryl, heteroaryl alkyl, and like moieties as framework andwith carboxyl, amine, hydroxyl, phenol, carbonyl, or thiol functionalgroups. Preferred synthetic amino acids include β-amino acids and homoor β analogs of natural amino acids.

[0150] Preferred framework amino acids include serine, threonine, ortyrosine, preferably serine or tyrosine, preferably tyrosine. FIG. 9illustrates serine as a framework for a building block and reactions forforming building blocks from serine, tyrosine, and other amino acids.Threonine and tyrosine typically exhibit reactivity similar to serine.Advantageously, serine, threonine, and tyrosine include: 1) multiple,orthogonal, well characterized reaction sites, 2) known methods andreactions for application as a combinatorial framework, 3) diversity ofsub-structures and domains which can be incorporated through thecarboxyl, α-amine, and hydroxyl functionalities, 4) compact distributionof the multiple reaction sites around a tetrahedral carbon framework,and 5) ready commercial availability of reagents for forming linkersand/or recognition elements.

[0151]FIG. 10 illustrates configurations in which recognition element,linker, and a chiral element can be coupled to a tyrosine framework.Threonine and serine can form analogous configurations. The chiralelement is a substituent that renders the carbon atom to which it isattached a chiral center. When one or more different recognitionelements are also substituents on or coupled to the chiral center, therecognition elements can adopt two or more enantiomeric configurations.Such enantiomers can be advantageous for providing diversity amongbuilding blocks.

[0152] Although not limiting to the present invention, a framework aminoacid, such as serine, threonine, or tyrosine, with a linker and tworecognition elements can be visualized with one of the recognitionelements in a pendant orientation and the other in an equatorialorientation, relative to the extended carbon chain of the framework.

[0153] Although not limiting to the present invention, the presentbuilding block framework can include: 1) diversity of framework reactionsites to maximize incorporation of potential receptor functionality, 2)reliable reaction and protection chemistries, 3) compact structure, 4)incorporation of diverse sub-structures, 5) a suitable platform forlinker element incorporation, and/or 6) development of non-equivalentdiversity domains to minimize redundancy in the receptor building blockswhile maximizing the number of functional groups and sub-structuresincorporated into a small library. Typically, the framework includesmultiple reaction sites with compact format. Compact format isadvantageous for providing a building block that fits at a suitabledensity on a support.

[0154] All of the naturally occurring and many synthetic amino acids arecommercially available. Further, forms of these amino acids derivatizedor protected to be suitable for reactions for coupling to recognitionelement(s) and/or linkers can be purchased or made by known methods(see, e.g., Green, TW; Wuts, PGM (1999), Protective Groups in OrganicSynthesis Third Edition, Wiley-Interscience, New York, 779 pp.;Bodanszky, M.; Bodanszky, A. (1994), The Practice of Peptide SynthesisSecond Edition, Springer-Verlag, New York, 217 pp.).

[0155] Preferred reaction schemes for preparing amino acids forreactions for forming building blocks according to the present inventioninclude those provided in the present Examples.

[0156] Recognition Element

[0157] The recognition element can be selected to provide one or morestructural characteristics to the building block. The framework caninteract with the ligand as part of the artificial receptor. Forexample, the recognition element can provide one or more structuralcharacteristics such as positive charge, negative charge, acid, base,electron acceptor, electron donor, hydrogen bond donor, hydrogen bondacceptor, free electron pair, π electrons, charge polarization,hydrophilicity, hydrophobicity, and the like. A recognition element canbe a small group or it can be bulky.

[0158] In an embodiment the recognition element can be a 1-12,preferably 1-6, preferably 1-4 carbon alkyl, substituted alkyl,cycloalkyl, heterocyclic, substituted heterocyclic, aryl alkyl, aryl,heteroaryl, heteroaryl alkyl, or like group. The recognition element canbe substituted with a group that includes or imparts positive charge,negative charge, acid, base, electron acceptor, electron donor, hydrogenbond donor, hydrogen bond acceptor, free electron pair, π electrons,charge polarization, hydrophilicity, hydrophobicity, and the like.

[0159] Recognition elements with a positive charge (e.g., at neutral pHin aqueous compositions) include amines, quaternary ammonium moieties,ferrocene, and the like. Suitable amines include alkyl amines, alkyldiamines, heteroalkyl amines, aryl amines, heteroaryl amines, aryl alkylamines, pyridines, heterocyclic amines (saturated or unsaturated, thenitrogen in the ring or not), amidines, hydrazines, and the like. Alkylamines generally have 1 to 12 carbons, preferably 1-8, rings can have3-12 carbons, preferably 3-8. Suitable alkyl amines include that offormula B9. Suitable heterocyclic or alkyl heterocyclic amines includethat of formula A9. Suitable pyridines include those of formulas A5 andB5. Any of the amines can be employed as a quaternary ammonium compound.Additional suitable quaternary ammonium moieties include trimethyl alkylquaternary ammonium moieties, dimethyl ethyl alkyl quaternary ammoniummoieties, dimethyl alkyl quaternary ammonium moieties, aryl alkylquaternary ammonium moieties, pyridinium quaternary ammonium moieties,and the like.

[0160] Recognition elements with a negative charge (e.g., at neutral pHin aqueous compositions) include carboxylates, phenols substituted withstrongly electron withdrawing groups (e.g., substitutedtetrachlorophenols), phosphates, phosphonates, phosphinates, sulphates,sulphonates, thiocarboxylates, and hydroxamic acids. Suitablecarboxylates include alkyl carboxylates, aryl carboxylates, and arylalkyl carboxylates. Suitable phosphates include phosphate mono-, di-,and tri-esters, and phosphate mono-, di-, and tri-amides. Suitablephosphonates include phosphonate mono- and di-esters, and phosphonatemono- and di-amides (e.g., phosphonamides). Suitable phosphinatesinclude phosphinate esters and amides.

[0161] Recognition elements with a negative charge and a positive charge(at neutral pH in aqueous compositions) include sulfoxides, betaines,and amine oxides.

[0162] Acidic recognition elements can include carboxylates, phosphates,sulphates, and phenols. Suitable acidic carboxylates includethiocarboxylates. Suitable acidic phosphates include the phosphateslisted hereinabove.

[0163] Basic recognition elements include amines. Suitable basic aminesinclude alkyl amines, aryl amines, aryl alkyl amines, pyridines,heterocyclic am ines (saturated or unsaturated, the nitrogen in the ringor not), amidines, and any additional amines listed hereinabove.Suitable alkyl amines include that of formula B9. Suitable heterocyclicor alkyl heterocyclic amines include that of formula A9. Suitablepyridines include those of formulas A5 and B5.

[0164] Recognition elements including a hydrogen bond donor includeamines, amides, carboxyls, protonated phosphates, protonatedphosphonates, protonated phosphinates, protonated sulphates, protonatedsulphinates, alcohols, and thiols. Suitable amines include alkyl amines,aryl amines, aryl alkyl amines, pyridines, heterocyclic amines(saturated or unsaturated, the nitrogen in the ring or not), amidines,ureas, and any other amines listed hereinabove. Suitable alkyl aminesinclude that of formula B9. Suitable heterocyclic or alkyl heterocyclicamines include that of formula A9. Suitable pyridines include those offormulas A5 and B5. Suitable protonated carboxylates, protonatedphosphates include those listed hereinabove. Suitable amides includethose of formulas A8 and B8. Suitable alcohols include primary alcohols,secondary alcohols, tertiary alcohols, and aromatic alcohols (e.g.,phenols). Suitable alcohols include those of formulas A7 (a primaryalcohol) and B7 (a secondary alcohol).

[0165] Recognition elements including a hydrogen bond acceptor or one ormore free electron pairs include amines, amides, carboxylates, carboxylgroups, phosphates, phosphonates, phosphinates, sulphates, sulphonates,alcohols, ethers, thiols, and thioethers. Suitable amines include alkylamines, aryl amines, aryl alkyl amines, pyridines, heterocyclic amines(saturated or unsaturated, the nitrogen in the ring or not), amidines,ureas, and amines as listed hereinabove. Suitable alkyl amines includethat of formula B9. Suitable heterocyclic or alkyl heterocyclic aminesinclude that of formula A9. Suitable pyridines include those of formulasA5 and B5. Suitable carboxylates include those listed hereinabove.Suitable amides include those of formulas A8 and B8. Suitablephosphates, phosphonates and phosphinates include those listedhereinabove. Suitable alcohols include primary alcohols, secondaryalcohols, tertiary alcohols, aromatic alcohols, and those listedhereinabove. Suitable alcohols include those of formulas A7 (a primaryalcohol) and B7 (a secondary alcohol). Suitable ethers include alkylethers, aryl alkyl ethers. Suitable alkyl ethers include that of formulaA6. Suitable aryl alkyl ethers include that of formula A4. Suitablethioethers include that of formula B6.

[0166] Recognition elements including uncharged polar or hydrophilicgroups include amides, alcohols, ethers, thiols, thioethers, esters,thio esters, boranes, borates, and metal complexes. Suitable amidesinclude those of formulas A8 and B8. Suitable alcohols include primaryalcohols, secondary alcohols, tertiary alcohols, aromatic alcohols, andthose listed hereinabove. Suitable alcohols include those of formulas A7(a primary alcohol) and B7 (a secondary alcohol). Suitable ethersinclude those listed hereinabove. Suitable ethers include that offormula A6. Suitable aryl alkyl ethers include that of formula A4.

[0167] Recognition elements including uncharged hydrophobic groupsinclude alkyl (substituted and unsubstituted), alkene (conjugated andunconjugated), alkyne (conjugated and unconjugated), aromatic. Suitablealkyl groups include lower alkyl, substituted alkyl, cycloalkyl, arylalkyl, and heteroaryl alkyl. Suitable lower alkyl groups include thoseof formulas A1, A3, and B1. Suitable aryl alkyl groups include those offormulas A3, A4, B3, and B4. Suitable alkyl cycloalkyl groups includethat of formula B2. Suitable alkene groups include lower alkene and arylalkene. Suitable aryl alkene groups include that of formula B4. Suitablearomatic groups include unsubstituted aryl, heteroaryl, substitutedaryl, aryl alkyl, heteroaryl alkyl, alkyl substituted aryl, andpolyaromatic hydrocarbons. Suitable aryl alkyl groups include those offormulas A3 and B4. Suitable alkyl heteroaryl groups include those offormulas A5 and B5.

[0168] Spacer recognition elements include hydrogen, methyl, ethyl, andthe like. Bulky recognition elements include 7 or more carbon or heteroatoms.

[0169] Formulas A1-A9 and B1-B9 are:

[0170] These A and B recognition elements can be called derivatives of,according to a standard reference: A1, ethylamine; A2, isobutylamine;A3, phenethylamine; A4, 4-methoxyphenethylamine;A5,2-(2-aminoethyl)pyridine; A6,2-methoxyethylamine; A7, ethanolamine;A8, N-acetylethylenediamine; A9,1-(2-aminoethyl)pyrrolidine; B 1, aceticacid, B2, cyclopentylpropionic acid; B3,3-chlorophenylacetic acid; B4,cinnamic acid; B5, 3-pyridinepropionic acid; B6, (methylthio)aceticacid; B7,3-hydroxybutyric acid; B8, succinamic acid; andB9,4-(dimethylamino)butyric acid.

[0171] In an embodiment, the recognition elements include one or more ofthe structures represented by formulas A1, A2, A3, A4, A5, A6, A7, A8,and/or A9 (the A recognition elements) and/or B11, B2, B3, B4, B5, B6,B7, B8, and/or B9 (the B recognition elements). In an embodiment, eachbuilding block includes an A recognition element and a B recognitionelement. In an embodiment, a group of 81 such building blocks includeseach of the 81 unique combinations of an A recognition element and a Brecognition element. In an embodiment, the A recognition elements arelinked to a framework at a pendant position. In an embodiment, the Brecognition elements are linked to a framework at an equatorialposition. In an embodiment, the A recognition elements are linked to aframework at a pendant position and the B recognition elements arelinked to the framework at an equatorial position.

[0172] Although not limiting to the present invention, it is believedthat the A and B recognition elements represent the assortment offunctional groups and geometric configurations employed by polypeptidereceptors. Although not limiting to the present invention, it isbelieved that the A recognition elements represent six advantageousfunctional groups or configurations and that the addition of functionalgroups to several of the aryl groups increases the range of possiblebinding interactions. Although not limiting to the present invention, itis believed that the B recognition elements represent six advantageousfunctional groups, but in different configurations than employed for theA recognition elements. Although not limiting to the present invention,it is further believed that this increases the range of bindinginteractions and further extends the range of functional groups andconfigurations that is explored by molecular configurations of thebuilding blocks.

[0173] Reagents that form many of the recognition elements arecommercially available. For example, reagents for forming recognitionelements A1, A2, A3, A4, A5, A6, A7, A8, A9 B1, B2, B3, B4, B5, B6, B7,B8, and B9 are commercially available.

[0174] Linkers

[0175] The linker is selected to provide a suitable covalent attachmentof the building block to a support. The framework can interact with theligand as part of the artificial receptor. The linker can also providebulk, distance from the support, hydrophobicity, hydrophilicity, andlike structural characteristics to the building block. Preferably, thelinker forms a covalent bond with a functional group on the framework.Preferably, before attachment to the support the linker also includes afunctional group that can be activated to react with or that will reactwith a functional group on the support. Preferably, once attached to thesupport, the linker forms a covalent bond with the support and with theframework.

[0176] The linker preferably forms or can be visualized as forming acovalent bond with an alcohol, phenol, thiol, amine, carbonyl, or likegroup on the framework. The linker can include a carboxyl, alcohol,phenol, thiol, amine, carbonyl, maleimide, or like group that can reactwith or be activated to react with the support. Between the bond to theframework and the group formed by the attachment to the support, thelinker can include an alkyl, substituted alkyl, cycloalkyl,heterocyclic, substituted heterocyclic, aryl alkyl, aryl, heteroaryl,heteroaryl alkyl, ethoxy or propoxy oligomer, a glycoside, or likemoiety.

[0177] The linker can include a good leaving group bonded to, forexample, an alkyl or aryl group. The leaving group being “good” enoughto be displaced by the alcohol, phenol, thiol, amine, carbonyl, or likegroup on the framework. Such a linker can include a moiety representedby the formula: R—X, in which X is a leaving group such as halogen(e.g., —Cl, Br or —I), tosylate, mesylate, triflate, and R is alkyl,substituted alkyl, cycloalkyl, heterocyclic, substituted heterocyclic,aryl alkyl, aryl, heteroaryl, heteroaryl alkyl, ethoxy or propoxyoligomer, a glycoside, or like moiety.

[0178] Preferred linker groups include those of formula: (CH₂)_(n)COOH,with n=1-16, preferably n=2-8, preferably n=2-6, preferably n=3.Reagents that form suitable linkers are commercially available andinclude any of a variety of reagents with orthogonal functionality.

[0179] Embodiments of Building Blocks

[0180] In an embodiment, building blocks can be represented by Formula2:

[0181] in which: RE₁ is recognition element 1, RE₂ is recognitionelement 2, and L is a linker. X is absent, C═O, CH₂, NR, NR₂, NH,NHCONH, SCONH, CH═N, or OCH₂NH. Preferably X is absent or C═O. Y isabsent, NH, O, CH₂, or NRCO. Preferably Y is NH or O. Preferably Y isNH. Z is CH₂, O, NH, S, CO, NR, NR₂, NHCONH, SCONH, CH═N, or OCH₂NH.Preferably Z is O. R₂ is H, CH₃, or another group that confers chiralityon the building block and has size similar to or smaller than a methylgroup. R₃ is CH₂; CH₂-phenyl; CHCH₃; (CH₂)_(n) with n=2-3; or cyclicalkyl with 3-8 carbons, preferably 5-6 carbons, phenyl, naphthyl.Preferably R₃ is CH₂ or CH₂-phenyl.

[0182] RE₁ is B1, B2, B3, B4, B5, B6, B7, B8, B9, A1, A2, A3, A4, A5,A6, A7, A8, or A9. Preferably RE₁ is B1, B2, B3, B4, B5, B6, B7, B8, orB9. RE₂ is A1, A2, A3, A4, A5, A6, A7, A8, A9, B1, B2, B3, B4, B5, B6,B7, B8, or B9. Preferably RE₂ is A1, A2, A3, A4, A5, A6, A7, A8, or A9.In an embodiment, RE₁ can be B2, B4, or B6 and RE₂ can be A2, A4, or A6.In an embodiment, RE₁ can be B1, B3, B6, or B8 and RE₂ can be A2, A4,A5, or A9. In an embodiment, RE₁ can be B2, B4, B6, or B8 and RE₂ can beA2, A4, A6, or A8. In an embodiment, RE₁ can be B1, B2, B4, B6, or B8and RE₂ can be A1, A2, A4, A6, or A8.

[0183] L is (CH₂)_(n)COOH, with n=1-16, preferably n=2-8, preferablyn=4-6, preferably n=3.

[0184] Embodiments of such building blocks include:

[0185] 4-{4-[(acetylamino-ethylcarbamoyl-methyl)-amino]-phenoxy}-butyricacid;

[0186]4-(4-{[(3-cyclopentyl-propionylamino)-ethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;

[0187]4-[4-({[2-(3-chloro-phenyl)-acetylamino]-ethylcarbamoyl-methyl}-amino)-phenoxy]-butyricacid;

[0188]4-(4-{[ethylcarbamoyl-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)-butyricacid;

[0189]4-(4-{[ethylcarbamoyl-(3-pyridin-3-yl-propionylamino)-methyl]-amino}-phenoxy)-butyricacid;

[0190]4-(4-{[ethylcarbamoyl-(2-methylsulfanyl-acetylamino)-methyl]-amino}-phenoxy)-butyricacid;

[0191]4-(4-{[ethylcarbamoyl-(3-hydroxy-butyrylamino)-methyl]-amino}-phenoxy)-butyricacid;

[0192]4-(4-{[(3-carbamoyl-propionylamino)-ethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;

[0193]4-(4-{[(4-dimethylamino-butyrylamino)-ethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;

[0194]4-{4-[(acetylamino-isobutylcarbamoyl-methyl)-amino]-phenoxy}-butyricacid;

[0195]4-(4-{[(3-cyclopentyl-propionylamino)-isobutylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;

[0196]4-[4-({[2-(3-chloro-phenyl)-acetylamino]-isobutylcarbamoyl-methyl}-amino)-phenoxy]-butyricacid;

[0197]4-(4-{[isobutylcarbamoyl-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)-butyricacid;

[0198]4-(4-{[isobutylcarbamoyl-(3-pyridin-3-yl-propionylamino)-methyl]-amino}-phenoxy)-butyricacid;

[0199]4-(4-{[isobutylcarbamoyl-(2-methylsulfanyl-acetylamino)-methyl]-amino}-phenoxy)-butyricacid;

[0200]4-(4-{[(3-hydroxy-butyrylamino)-isobutylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;

[0201]4-(3-{[(3-carbamoyl-propionylamino)-isobutylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;

[0202]4-(4-{[(4-dimethylamino-butyrylamino)-isobutylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;

[0203]4-{4-[(acetylamino-phenethylcarbamoyl-methyl)-amino]-phenoxy}-butyricacid;

[0204]4-(4-{[(3-cyclopentyl-propionylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;

[0205]4-[4-({[2-(3-chloro-phenyl)-acetylamino]-phenethylcarbamoyl-methyl}-amino)-phenoxy]-butyricacid;

[0206]4-(4-{[phenethylcarbamoyl-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)-butyricacid;

[0207]4-(4-{[phenethylcarbamoyl-(3-pyridin-3-yl-propionylamino)-methyl]-amino}-phenoxy)-butyricacid;

[0208]4-(4-{[(2-methylsulfanyl-acetylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;

[0209]4-(4-{[(3-hydroxy-butyrylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;

[0210]4-(4-{[(3-carbamoyl-propionylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;

[0211]4-(4-{[(4-dimethylamino-butyrylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)butyricacid;

[0212]4-[4-({acetylamino-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}-amino)-phenoxy]butyricacid;

[0213]4-[4-({(3-cyclopentyl-propionylamino)-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}amino)-phenoxy]-butyricacid;

[0214]4-[4-({[2-(3-chloro-phenyl)-acetylamino]-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}amino)-phenoxy]-butyricacid;

[0215]4-(4-{[[2-(4-methoxy-phenyl)-ethylcarbamoyl]-(3-phenyl-acryloylamino)-methyl]-amino}phenoxy)-butyricacid;

[0216]4-(4-{[[2-(4-methoxy-phenyl)-ethylcarbamoyl]-(3-pyridin-3-yl-propionylamino)-methyl]amino}-phenoxy)-butyricacid;

[0217]4-(4-{[[2-(4-methoxy-phenyl)-ethylcarbamoyl]-(2-methylsulfanyl-acetylamino)-methyl]amino}-phenoxy)-butyricacid;

[0218]4-[4-({(3-hydroxy-butyrylamino)-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}-amino)phenoxy]-butyricacid;

[0219]4-[4-({(3-carbamoyl-propionylamino)-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}amino)-phenoxy]-butyricacid;

[0220]4-[4-({(4-dimethylaino-butyrylamino)-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}-amino)-phenoxy]-butyricacid;

[0221]4-(4-{[acetylamino-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;

[0222]4-(4-{[(3-cyclopentyl-propionylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;

[0223]4-(4-{[[2-(3-chloro-phenyl)-acetylamino]-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0224]4-(4-{[(3-phenyl-acryloylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;

[0225]4-(4-{[(2-pyridin-2-yl-ethylcarbamoyl)-(3-pyridin-3-yl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;

[0226]4-(4-{[(2-methylsulfanyl-acetylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;

[0227]4-(4-{[(3-hydroxy-butyrylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;

[0228]4-(4-{[(3-carbamoyl-propionylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0229]4-(4-{[(4-dimethylamino-butyrylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0230]4-(4-{[acetylamino-(2-methoxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;

[0231]4-(4-{[(3-cyclopentyl-propionylamino)-(2-methoxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0232]4-(4-{[[2-(3-chloro-phenyl)-acetylamino]-(2-methoxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0233]4-(4-{[(2-methoxy-ethylcarbamoyl)-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)butyricacid;

[0234]4-(4-{[(2-methoxy-ethylcarbamoyl)-(3-pyridin-3-yl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;

[0235]4-(4-{[(2-methoxy-ethylcarbamoyl)-(2-methylsulfanyl-acetylamino)-methyl]-amino}phenoxy)-butyric acid;

[0236]4-(4-{[(3-hydroxy-butyrylamino)-(2-methoxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;

[0237]4-(3-{[(3-carbamoyl-propionylamino)-(2-methoxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;

[0238]4-(4-{[(4-dimethylamino-butyrylamino)-(2-methoxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;

[0239]4-(4-{[acetylamino-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;

[0240]4-(4-{[(3-cyclopentyl-propionylamino)-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0241]4-(4-{[[2-(3-chloro-phenyl)-acetylamino]-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0242]4-(4-{[(2-hydroxy-ethylcarbamoyl)-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)butyricacid;

[0243]4-(4-{[(2-hydroxy-ethylcarbamoyl)-(3-pyridin-3-yl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;

[0244]4-(4-{[(2-hydroxy-ethylcarbamoyl)-(2-methylsulfanyl-acetylamino)-methyl]-amino}-phenoxy)butyricacid;

[0245]4-(4-{[(3-hydroxy-butyrylamino)-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;

[0246]4-(3-{[(3-carbamoyl-propionylamino)-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;

[0247]4-(4-{[(4-dimethylamino-butyrylamino)-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0248]4-(4-{[acetylamino-(2-acetylamino-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;

[0249]4-(4-{[(2-acetylamino-ethylcarbamoyl)-(3-cyclopentyl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;

[0250]4-[4-({(2-acetylamino-ethylcarbamoyl)-[2-(3-chloro-phenyl)-acetylamino]-methyl}-amino)phenoxy]-butyricacid;

[0251]4-(4-{[(2-acetylamino-ethylcarbamoyl)-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)butyricacid;

[0252]4-(4-{[(2-acetylamino-ethylcarbamoyl)-(3-pyridin-3-yl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;

[0253]4-(4-{[(2-acetylamino-ethylcarbamoyl)-(2-methylsulfanyl-acetylamino)-methyl]-amino}-phenoxy)-butyricacid;

[0254]4-(4-{[(2-acetylamino-ethylcarbamoyl)-(3-hydroxy-butyrylamino)-methyl]-amino}-phenoxy)butyricacid;

[0255]4-(3-{[(2-acetylamino-ethylcarbamoyl)-(3-carbamoyl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;

[0256]4-(4-{[(2-acetylamino-ethylcarbamoyl)-(4-dimethylamino-butyrylamino)-methyl]-amino}-phenoxy)-butyricacid;

[0257]4-(4-{[acetylamino-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;

[0258]4-(4-{[(3-cyclopentyl-propionylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0259]4-(4-{[[2-(3-chloro-phenyl)-acetylamino]-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0260]4-(4-{[(3-phenyl-acryloylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0261]4-(4-{[(3-pyridin-3-yl-propionylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0262]4-(4-{[(2-methylsulfanyl-acetylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0263]4-(4-{[(3-hydroxy-butyrylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0264]4-(3-{[(3-carbamoyl-propionylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0265]4-(4-{[(4-dimethylamino-butyrylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;

[0266] salts thereof, esters thereof, protected or blocked derivativesthereof, immobilized derivatives thereof, derivatives thereof, ormixtures thereof. The nomenclature in this paragraph is according to theprogram CS CHEMDRAW ULTRA®.

[0267] Building blocks of Formula 2 and including an A recognitionelement, a B recognition element, a linker, and a framework of anaturally occurring α-amino acid can be visualized as having the Brecognition element in an equatorial configuration and the A recognitionelement in a pendant configuration. An embodiment of such aconfiguration is schematically illustrated in Scheme 3:

[0268] Building blocks including an A and/or a B recognition element, alinker, and an amino acid framework can be made by methods illustratedin general Scheme 4.

[0269] More on Building Blocks

[0270] Building blocks can be asymmetric. Employing asymmetry, variouscombinations of, for example, linker and recognition elements canproduce building blocks that can be visualized to occupy 3D space indifferent ways. As a consequence, these different building blocks canperform binding related but otherwise distinct functions.

[0271] In an embodiment, building blocks including two recognitionelements, a linker, and a framework can be visualized as having bothrecognition elements in spreading pendant configurations. An embodimentof such a configuration is schematically illustrated in Scheme 5:

[0272] Such a configuration has a molecular footprint with substantialarea in two dimensions. Such a larger footprint can be suitable, forexample, for binding larger ligands that prefer or require interactionswith a receptor over a larger area or that prefer or requireinteractions with a larger number of functional groups on therecognition element. Such larger ligands can include proteins,carbohydrates, cells, and microorganisms (e.g., bacteria and viruses).

[0273] In an embodiment, a building block can have only a singlerecognition element in a pendant configuration and a pendant linkerdistal on the framework. Such building blocks can be compact. Such abuilding block can interact with large molecules that include a bindingregion, such as a protein (e.g., enzyme or receptor) or othermacromolecule. For example, such a building block can be employed toprobe cavities, such as binding sites, on proteins.

[0274] Sets of Building Blocks

[0275] The present invention also relates to sets of building blocks.The sets of building blocks can include isolated building blocks,building blocks with an activated linker for coupling to a support,and/or building blocks coupled to a support. Sets of building blocksinclude a plurality of building blocks. The plurality of building blockscan be a component of a coating, of a spot or spots (e.g., formingcandidate artificial receptor(s)), or of a kit. The plurality ofbuilding blocks can include a sufficient number of building blocks andrecognition elements for exploring candidate artificial receptors or fordefining receptors for a ligand. That is, the set of building blocks caninclude a majority (preferably at least 6) of the structuralcharacteristics selected from positive charge, negative charge, acid,base, electron acceptor, electron donor, hydrogen bond donor, hydrogenbond acceptor, free electron pair, π electrons, charge polarization,hydrophilicity, hydrophobicity.

[0276] For a set of building blocks, the recognition elements arepreferably selected to provide a variety of structural characteristicsto the individual members of the set. A single building block caninclude recognition elements with more thah one of the structuralcharacteristics. A set of building blocks can include recognitionelements with each of the structural characteristics. For example, a setof building blocks can include one or more building blocks including apositively charged recognition element, one or more building blocksincluding a negatively charged recognition element, one or more buildingblocks including an acidic recognition element, one or more buildingblocks including a basic recognition element, one or more buildingblocks including an electron donating recognition element, one or morebuilding blocks including an electron accepting recognition element, oneor more building blocks including a hydrogen bond donor recognitionelement, one or more building blocks including a hydrogen bond acceptorrecognition element, one or more building blocks including a polarrecognition element, one or more building blocks including a recognitionelement with free electron pair(s), one or more building blocksincluding a recognition element with π electrons, one or more buildingblocks including a hydrophilic recognition element, one or more buildingblocks including a hydrophobic recognition element, one or more buildingblocks including a small recognition element, and/or one or morebuilding blocks including a bulky recognition element.

[0277] In an embodiment, the number and variety of recognition elementsis selected to provide a set of building blocks with a manageable numberof members. A manageable number of building blocks provides, typically,fewer than 10 million combinations, preferably about 2 millioncombinations, with each combination including, preferably, 3, 4, 5, or 6building blocks. In an embodiment, the recognition elements provide aset of building blocks that incorporate the functional groups andconfigurations found in the components of natural receptors, preferablywith the smallest number of building blocks.

[0278] The nine A and nine B recognition elements can be incorporatedinto a set of 81 (9×9) building blocks, each with one A and one Brecognition element. Such building blocks can, for example, be preparedusing combinatorial syntheses on a framework, such as a serine ortyrosine framework. In groups of 4, this set of 81 building blocksprovides 1.66 million combinations of building blocks (Table 1), each ofwhich can be a heterogeneous combination in a microarray on a support,substrate, or scaffold. Although not limiting to the present invention,it is believed that these groups of 4 are sufficient to incorporate thefunctional groups and configurations found in natural receptors and toprovide sufficient candidate artificial receptors to yield one or moreartificial receptors for a specified ligand. TABLE 1 Calculation of theNumber of Candidate Artificial Receptor Combinations Discretecombinations calculated using the following formula for N compoundstaken in groups of n (CRC Standard Math Tables and Formulas Handbook,30th ed.): Number of Combinations = N!/(N-n)! n! For N = 81 GROUPCOMBINATIONS n = 1 81 n = 2 3,240 n = 3 85,320 n = 4 1,663,740

[0279] A set of building blocks can include building blocks of generalFormula 2, with RE₁ being B1, B2, B3, B4, B5, B6, B7, B8, or B9 and withRE₂ being A1, A2, A3, A4, A5, A6, A7, A8, or A9. In an embodiment of theset, RE₁ can be B2, B4, or B6 and RE₂ can be A2, A4, or A6. In anembodiment of the set, RE₁ can be B1, B3, B6, or B8 and RE₂ can be A2,A4, A5, or A9. In an embodiment of the set, RE₁ can be B2, B4, B6, or B8and RE₂ can be A2, A4, A6, or A8. In an embodiment of the set, RE₁ canbe B1, B2, B4, B6, or B8 and RE₂ can be A1, A2, A4, A6, or A8. In anembodiment of the set, RE₁ can be B1, B2, B3, B4, B5, B6, B7, B8, or B9and RE₂ can be A1, A2, A3, A4, A5, A6, A7, A8, or A9.

[0280] In an embodiment, a set of building blocks includes alkyl, aryl,and polar recognition elements, plus recognition elements that arecombinations of these structural characteristics. A set of buildingblocks including those of general Formula 2, with RE₁ being B1, B2, B3,B4, B5, B6, B7, B8, or B9 and with RE₂ being A1, A2, A3, A4, A5, A6, A7,A8, or A9 is a set of building blocks with includes alkyl, aryl, andpolar recognition elements. Table 2 illustrates an embodiment of 81building blocks of general Formula 2 with recognition elements that spanalkyl, aryl, and polar recognition elements. TABLE 2 Embodiment of 81Building Blocks of General Formula 2 with Recognition Elements that SpanAlkyl, Aryl, and Polar Recognition Elements. RE₁, EQUATORIAL RE1 RE2 B1B2 B3 B4 B5 B6 B7 B8 B9 RE₂ A1 A1-B1 A1-B2 A1-B3 A1-B4 A1-B5 A1-B6 A1-B7A1-B8 A1-B9 PENDANT A2 A2-B1 A2-B2 A2-B3 A2-B4 A2-B5 A2-B6 A2-B7 A2-B8A2-B9 A3 A3-B1 A3-B2 A3-B3 A3-B4 A3-B5 A3-B6 A3-B7 A3-B8 A3-B9 A4 A4-B1A4-B2 A4-B3 A4-B4 A4-B5 A4-B6 A4-B7 A4-B8 A4-B9 A5 A5-B1 A5-B2 A5-B3A5-B4 A5-B5 A5-B6 A5-B7 A5-B8 A5-B9 A6 A6-B1 A6-B2 A6-B3 A6-B4 A6-B5A6-B6 A6-B7 A6-B8 A6-B9 A7 A7-B1 A7-B2 A7-B3 A7-B4 A7-B5 A7-B6 A7-B7A7-B8 A7-B9 A8 A8-B1 A8-B2 A8-B3 A8-B4 A8-B5 A8-B6 A8-B7 A8-B8 A8-B9 A9A9-B1 A9-B2 A9-B3 A9-B4 A9-B5 A9-B6 A9-B7 A9-B8 A9-B9

[0281] Embodiments of Sets of Building Blocks

[0282] The present invention includes sets of building blocks. Sets ofbuilding blocks can include 2 or more building blocks coupled to asupport or scaffold. Such a support or scaffold can be referred to asincluding heterogeneous building blocks. As used herein, the term“support” refers to a solid support that is, typically, macroscopic. Asused herein, the term scaffold refers to a molecular scale structure towhich a plurality of building blocks can covalently bind. The two ormore building blocks can be coupled to the support or scaffold in amolecular configuration with different building blocks in proximity toone another. Such a molecular configuration of a plurality of differentbuilding blocks provides a candidate artificial receptor.

[0283] Building Blocks on Supports

[0284] The present invention includes immobilized sets and combinationsof building blocks. In an embodiment, the present invention includes asolid support having on its surface a plurality of building blocks.

[0285] For example, the support can be a glass tube or well coated witha plurality of building blocks. In an embodiment, the surface of theglass tube or well (e.g., a 96 well plate) coated with a coating towhich the plurality of building blocks are covalently bound. Such acoating can be referred to as including heterogeneous building blocks.The surface or coating can include a density of building blockssufficient to provide interactions of more than one building block witha ligand. The building blocks can be in proximity to one another.Evidence of proximity of different building blocks is provided byaltered (e.g., tighter or looser) binding of a ligand to a surface witha plurality of building blocks compared to a surface with only one ofthe building blocks.

[0286] A set of building blocks can be employed in combinations of 2, 3,4, or more building blocks on an individual tube or well. For thisembodiment, with each combination using a bulky tube or well, amanageable set of building blocks preferably provides fewer than severalhundred or several thousand combinations of building blocks. Forexample, in this context, a set of 3, 4, 5, or 6 building blocksprovides a manageable number of combinations of 2, 3, or 4 buildingblocks.

[0287] In an embodiment, immobilized combinations of building blocks caninclude a plurality of tubes each tube having immobilized on its surfacea heterogeneous combination of building blocks. The building blocks canbe immobilized on the surface of the tube through amide links betweeneach building block and a support matrix. The immobilized buildingblocks can include combinations of 2, 3, or 4 building blocks. Forconvenience in limiting the number of tubes handled, in this embodimenta set includes up to 5-7 building blocks, preferably 5 or fewer,preferably 3, 4, or 5. For tubes, suitable building blocks have generalFormula 2, with RE₁ being B1, B2, B3, B4, B5, B6, B7, B8, or B9 and withRE₂ being A1, A2, A3, A4, A5, A6, A7, A8, or A9. In an embodiment fortubes, RE₁ can be B1, B3, B6, or B8 and RE₂ can be A2, A4, A5, or A9. Inan embodiment for tubes, RE₁ can be B2, B4, or B6 and RE₂ can be A2, A4,or A6. In an embodiment for tubes, RE₁ can be B2, B4, B6, or B8 and RE₂can be A2, A4, A6, or A8. In an embodiment for tubes, RE₁ can be B1, B2,B4, B6, or B8 and RE₂ can be A1, A2, A4, A6, or A8. A plurality of tubeseach coated with a combination of building blocks can be configured asan array of tubes.

[0288] In an embodiment, the present invention includes a solid supporthaving on its surface a plurality of regions or spots, each region orspot including a plurality of building blocks. For example, the supportcan be a glass slide spotted with a plurality of spots, each spotincluding a plurality of building blocks. Such a spot or region can bereferred to as including heterogeneous building blocks. Each region orspot can include a density of building blocks sufficient to provideinteractions of more than one building block with a ligand. Althougheach region or spot is typically separated from the others, in theregion or spot, the building blocks can be in proximity to one another.Evidence of proximity of different building blocks in a region or spotis provided by altered (e.g., tighter or looser) binding of a ligand toa surface with a plurality of building blocks compared to a region orspot with only one of the building blocks. A plurality of regions orspots of building blocks is referred to herein as an array of regions orspots.

[0289] A set of building blocks can be employed in combinations of 2, 3,4, or more building blocks in each region or spot. In such anembodiment, up to 100,000 spots can fit on a glass slide. Therefore, amanageable set of building blocks can provide several millioncombinations of building blocks. For example, in this context, a set of81 building blocks provides a manageable number of (1.66 million)combinations of 4 building blocks. Although not limiting to the presentinvention, it is believed that these 1.66 million combinations aresufficient to incorporate the functional groups and configurations foundin natural receptors and to provide sufficient candidate artificialreceptors to yield one or more artificial receptors for a specifiedligand.

[0290] In an embodiment, immobilized combinations of building blocks caninclude one or more glass slides, each slide having on its surface aplurality of spots, each spot including an immobilized heterogeneouscombination of building blocks. The building blocks can be immobilizedon the surface of the slide through amide links between each buildingblock and a support matrix. The immobilized building blocks can include,for example, combinations of 2, 3, 4, 5, or 6 building blocks.

[0291] For convenience in limiting the number of slides handled, in thisembodiment a set includes up to 200 building blocks, preferably 50-100,preferably about 80 (e.g., 81) building blocks. For slides, suitablebuilding blocks have general Formula 2, with RE₁ being B 1, B2, B3, B4,B5, B6, B7, B8, or B9 and with RE₂ being A1, A2, A3, A4, A5, A6, A7, A8,or A9. This embodiment can include a group of slides with 1.7 millionheterogeneous spots, each spot including 4 building blocks.

[0292] In an embodiment, the one or more slides can includeheterogeneous spots of building blocks made from combinations of asubset of the total building blocks and/or smaller groups of thebuilding blocks in each spot. That is, each spot includes only, forexample, 2 or 3 building blocks, rather than 4 or 5. For example, theone or more slides can include the number of spots formed bycombinations of a full set of building blocks (e.g. 81 of a set of 81)in groups of 2 and/or 3. For example, the one or more slides can includethe number of spots formed by combinations of a subset of the buildingblocks (e.g., 25 of the set of 81) in groups of 4 or 5. For example, theone or more slides can include the number of spots formed bycombinations of a subset of the building blocks (e.g., 25 of the set of81) in groups of 2 or 3. Should a candidate artificial receptor ofinterest be identified from the subset and/or smaller groups, thenadditional subsets and groups can be made or selected incorporating thebuilding blocks in the candidates of interest or structurally similarbuilding blocks.

[0293] For example, FIG. 7 illustrates that a single slide with the3,240 n=2 derived combinations can be used to define a more limited setfrom the 81 building blocks. This defined set of e.g. 25 (defined from a5×5 matrix of the n=2 results) can be used to produce an additional2,300 n=3 derived and 12,650 n=4 derived combinations which can beprobed to define the optimum receptor configuration. Furtheroptimization can be pursued using ratios of the best building blockswhich deviate from 1:1 followed by specific synthesis of the identifiedreceptor(s).

[0294] Building blocks can be coupled to supports using known methodsfor activating compounds of the types employed as building blocks andfor coupling them to supports. For example, building blocks includingactivated esters can be coupled to supports including amine functionalgroups. A carboxyl group on a building block can be derivatized to formthe activated ester. By way of further example, building blocksincluding amine functional groups can be coupled to supports includingcarboxyl groups. Pairs of functional groups that can be employed onbuilding blocks and supports include amine and carboxyl (or activatedcarboxyl), thiol and maleimide, and the like.

[0295] Individual or combinations of building blocks can be coupled tothe supports in spots using conventional micro spotting techniques(e.g., piezoelectric, pin, and electromagnetic printers). Such spottingyields a microarray of spots of heterogeneous combinations of buildingblocks, each of which can be a candidate artificial receptor. Asdescribed herein above, each spot in a microarray includes astatistically significant number of each building block.

[0296] The set of building blocks can be on any of the variety of knownsupports employed in combinatorial or synthetic chemistry (e.g., amicroscope slide, a bead, a resin, a gel, or the like). Suitablesupports include functionalized glass, such as a functionalized slide ortube, glass microscope slide, glass plate, glass coverslip, glass beads,microporous glass beads, silica gel supports, and the like. As describedhereinabove, a glass support can include a support matrix of silanatingagent with functional groups suitable for coupling to a building block.For use in sets of building blocks, the support matrix functional groupscan be pendant from the support in groups of one (e.g., as a lawn ofamines or another functional group) or in groups of, for example, 2, 3,4, 5, 6, or 7. The groups of a plurality of functional groups pendantfrom the support can be visualized as scaffold molecules pendant fromthe support.

[0297] The surface of the support can be visualized as including a floorand the building blocks (FIGS. 3A, 3B, and 4). Thus, the floor can beconsidered a feature of the candidate artificial receptor. In anembodiment, the candidate artificial receptor can include buildingblocks and unmodified amines of the floor. Such a candidate artificialreceptor has an amine/ammonium floor. In an embodiment, the candidateartificial receptor can include building blocks and modified amines ofthe floor (e.g., the acetamide).

[0298] Sets on Scaffolds

[0299] In an embodiment, the present invention includes a scaffoldmolecule having coupled to it a plurality of building blocks. Forexample, the scaffold can be a polyamine, for example, a cyclic moleculewith a plurality of primary amine groups around the ring. Such ascaffold can include a plurality of building blocks coupled to theamines. Such a scaffold can be referred to as including heterogeneousbuilding blocks. The scaffold can provide a density of building blockssufficient to provide interactions of more than one building block witha ligand. The building blocks can be in proximity to one another.Evidence of proximity of different building blocks on a scaffold isprovided by altered (e.g., tighter or looser) binding of a ligand to ascaffold with a plurality of building blocks compared to the scaffoldwith only one of the building blocks. The scaffold can be coupled to asupport. Scaffolds can include functional groups for coupling to, forexample, 2, 3, 4, 5, 6, or 7 building blocks.

[0300] A scaffold can be the support for an artificial receptorincluding a combination of 3, 4, or more building blocks occupyingdistinct positions relative to one another on the scaffold. For example,building block 1 can be adjacent to any of building blocks 2, 3, or 4.This can be illustrated by considering the building blocks coupled todifferent functional groups on a scaffold. For example, FIG. 8illustrates positional isomers of 4 different building blocks at thevertices of a quadrilateral shaped scaffold. Scaffold positional isomerartificial receptors can be made, for example, on a scaffold withmultiple functional groups that can be protected and deprotected byorthogonal chemistries.

[0301] Such a scaffold positional isomer artificial receptor can providea lead or working receptor with utility distinct from a solid supportbased receptor. For example, such a scaffold positional isomer can beevaluated and selected for optimal binding, then employed where anoptimal receptor is required. The scaffold artificial receptor can beimmobilized, for example, on a light fiber to provide a detectablesignal or for any of the other applications described herein for workingartificial receptors.

[0302] A scaffold artificial receptor that has not been immobilized canbe used in applications in which an antibody can be used, as a specificanticancer agent, to bind and immobilize/neutralize bloodstreamcomponents like cholesterol, cocaine or DDT, to bind and neutralizehazardous wastes, in the development of free solution analysis methods,e.g. fluorescence polarization immunoassay or molecular beacon basedassays. Such free (not immobilized) scaffold artificial receptors canalso be used for development of pharmaceuticals based on binding, e.g.application of scaffold receptors to block protein-protein interactionswhich are involved in cancer, the progression of AIDS, the developmentof tuberculoses and malaria, the toxic effects produced by exposure toindustrial chlorinated aromatics, and the like.

[0303] In an embodiment, the scaffold artificial receptor is introducedinto a subject (e.g., mouse, rat, dog, cat, horse, monkey, human, or thelike) through, for example, injection, ingestion, gavage, suppository,inhalation, or the like. Once introduced, the scaffold artificialreceptor can bind a compound of interest, such as cocaine, cholesterol,lead, DDT. Binding of the scaffold artificial receptor binding cantarget the bound material for detection, destruction, excretion,therapy, or the like.

[0304] In an embodiment, the scaffold artificial receptor is contactedwith an environmental matrix (e.g., water, soil, sediment) through, forexample, mixing, spraying, injection, or the like. In the matrix, thescaffold artificial receptor binds a ligand of interest. For a ligand ofinterest that is a hazardous waste component, a hazardous waste mixture,a pollution component, a pollution mixture, or the like, binding to thescaffold artificial receptor can target the bound material fordetection, destruction, or immobilization.

[0305] In an embodiment, the scaffold artificial receptor is to aconjugated biological effector. Such a biological effector can be atoxin, a radioisotope chelate, or the like. The conjugate can beintroduced into a subject. After introduction, the scaffold artificialreceptor conjugate can interact with a ligand of interest that isassociated with, for example, a disease causing microbe or a cancercell. This interaction targets the conjugated toxin or radioisotopechelate to the disease causing microbe or cancer cell for the detection,therapy, destruction of the infectious agent or cancerous cell.

[0306] In an embodiment, the scaffold artificial receptor is used infree solution analysis methods. For example a scaffold artificialreceptor can include a fluorophore or molecular beacon. Binding of thescaffold artificial receptor conjugate to a ligand of interest or asample containing a ligand of interest then produces fluorescencepolarization or molecular beacon recombination which produces a signalwhich is related to the presence of the ligand of interest.

[0307] In an embodiment, the scaffold artificial receptor can be used asa pharmaceutical, for example, for the treatment of cancer, infection,disease, or toxic effects. As a pharmaceutical, binding of the scaffoldartificial receptor to a ligand of interest (e.g., on or in a cell ormicrobe) can block, for example, DNA replication, gene regulation, RNAtranscription, peptide synthesis. Such blocking can disrupt protein(e.g., enzyme) synthesis or modification, protein-protein interactionsor the like. Such synthesis, modification, or interactions can beinvolved in cancer, HIV/AIDS, tuberculosis, malaria, or the toxiceffects produced by exposure to industrial chlorinated aromatics or thelike. Thus, the scaffold artificial receptor can treat these disorders.

[0308] The scaffold molecule can be any of the variety of knownmolecular scaffolds employed in combinatorial research. Suitablescaffold molecules include those illustrated in Scheme 6. The compoundsillustrated in Scheme 6 are either commercially available or can be madeby known methods. For example, compounds 1, 2, 4, and 5 are commerciallyavailable from Aldrich. Compound 3 can be prepared by the method ofPattarawarapan (2000) (Pattarawarapan, M and Burgess, K, “A LinkerScaffold to Present Dimers of Pharmacophores Prepared by Solid-PhaseSynthesis”, Angew. Chem. Int. Ed., 39, 4299-4301 (2000)). Compound 6 canbe made in the o-NH₂ form (shown) by the method of Kimura (2001)(Kimura, M; Shiba, T; Yamazaki, M; Hanabusa, K; Shirai, H and Kobayashi,N, “Construction of Regulated Nanospace around a Porphyrin Core”, J. Am.Chem. Soc., 123, 5636-5642 (2001)) and in the p-COOH (not shown) by themethod of Jain (2000) (Jain, RK; Hamilton, AD (2000), “Protein SurfaceRecognition by Synthetic Receptors Based on a TetraphenylporphyrinScaffold”, Org. Lett. 2, pp. 1721-1723). Compound 7 can be made in the—COOH form (shown) or in the —OH form (not shown) by the method ofHamuro (1997) (Hamuro, Y. et al., (Andrew Hamilton), “A Calixarene withfour Peptide Loops: An Antibody Mimic for Recognition of ProteinSurfaces”, Angew. Chem. Int. Ed. Engl., 36, pp. 2680-2683). Compound 8can be used with three functional groups in the —NH₂ form (shown), withfour functional groups including both the —COOH and —NH₂ groups (asshown), or as a dimer product with 6 —NH₂ functional groups (not shown).Each of these forms of compound 8 can be made by the method of Opatz(2001) (Opatz, T; Liskamp, RM (2001), “A Selectively DeprotectableTriazacyclophane Scaffold for the Construction of Artificial Receptors”,Org. Lett., 3, pp. 3499-3502).

[0309] Molecular Configurations in Combinations of Building Blocks

[0310]FIG. 11 schematically illustrates a molecular configuration ofbuilding blocks that can provide a region for binding for a smallmolecule ligand. FIG. 11 illustrates that a plurality of adjacentbuilding blocks, each with a pendant and an equatorial recognitionelement, can form a cavity or other binding site. The binding site canbe sized to serve as a receptor for, for example, a small moleculeligand of interest. Space filling molecular models of embodiments ofbuilding blocks can be envisioned to fit this schematic. Neighboringbuilding blocks that are different from one another can providediversity to the binding interactions available in the binding site.

[0311]FIG. 12 schematically illustrates a molecular configuration ofbuilding blocks that can provide a broad binding site with a largesurface area. FIG. 12 illustrates that a plurality of adjacent buildingblocks, each with two pendant lateral recognition elements, can form abroad binding site with a large molecular footprint. The broad bindingsite can serve as a receptor for, for example, a macromolecule ligand ofinterest, a cell, or a microorganism (e.g., a bacterium or a virus).Space filling molecular models of embodiments of building blocks can beenvisioned to fit this schematic. Neighboring building blocks that aredifferent from one another can provide diversity to the bindinginteractions available in the binding site.

[0312]FIG. 13 schematically illustrates a molecular configuration ofbuilding blocks arranged to form a protruding binding site, which can,for example, bind a test ligand with a cavity. FIG. 13 illustrates thata plurality of adjacent building blocks, each with a pendant protrudingrecognition element, can form a protruding binding site. The protrudingbinding site can serve as a receptor for, for example, a macromoleculehaving an active or binding site. Space filling molecular models ofembodiments of building blocks can be envisioned to fit this schematic.Neighboring building blocks that are different from one another canprovide diversity to the binding interactions available in the bindingsite. The binding site can include recognition elements from 2 or morebuilding blocks.

[0313]FIG. 8 illustrates that a molecular configuration of buildingblocks can form 6 positional isomers. This illustration places thebuilding blocks at corners of a square, but the same is true of 4vertices of any quadrilateral. Candidate or lead artificial receptorshaving the structure of the different positional isomers can be made ona scaffold.

[0314] Embodiments of Sets as Reagents

[0315] The present invention includes sets of building blocks asreagents. Reagent sets of building blocks can include individual ormixtures of building blocks. The reagent sets can be used to makeimmobilized building blocks and groups of building blocks, and can besold for this purpose. In an embodiment, the set includes buildingblocks with recognition elements representing hydrophobic alkyl,hydrophobic aryl, hydrogen bond acceptor, basic, hydrogen bond donor,and small size as structural characteristics. For example, the set caninclude building blocks of general Formula 2, with RE₁ being B1, B2, B3,B4, B5, B6, B7, B8, or B9 and with RE₂ being A1, A2, A3, A4, A5, A6, A7,A8, or A9. In an embodiment of the set, RE₁ can be B1, B3, B6, or B8 andRE₂ can be A2, A4, A5, or A9. In an embodiment of the set, RE₁ can beB2, B4, or B6 and RE₂ can be A2, A4, or A6. In an embodiment of the set,RE₁ can be B2, B4, B6, or B8 and RE₂ can be A2, A4, A6, or A8. In anembodiment of the set, RE₁ can be B1, B2, B4, B6, or B8 and RE₂ can beA1, A2, A4, A6, or A8. In an embodiment of the kit, RE₁ can be B1, B2,B3, B4, B5, B6, B7, B8, or B9 and RE₂ can be A1, A2, A3, A4, A5, A6, A7,A8, or A9. The building blocks can include as L (CH₂)_(n)COOH, withn=1-16, preferably n=2-8, preferably n=4-6, preferably n=3, or anactivated form of L, for example, an activated ester.

[0316] The set can be part of a kit including containers of one ormixtures of building blocks, the containers can be in a package, and thekit can include written material describing the building blocks andproviding instructions for their use.

[0317] The present invention may be better understood with reference tothe following examples. These examples are intended to be representativeof specific embodiments of the invention, and are not intended aslimiting the scope of the invention.

EXAMPLES Example 1 Synthesis of Building Blocks

[0318] Selected building blocks representative of thealkyl-aromatic-polar span of the entire building block grid of Table 2(above) were synthesized for demonstrating effectiveness of thesebuilding blocks for making candidate artificial receptors. Thesebuilding blocks were made on a framework of general Formula 2,specifically tyrosine, and included recognition element pairs A2B2,A4B4, and A6B6. These recognition element pairs were selected along thediagonal of Table 2, and include enough of the range from alkyl, toaromatic, to polar to represent a significant degree of the interactionsand functional groups of the full set of 81 such building blocks.

[0319] This selected group of building blocks (N=3) was employed todemonstrate synthesis, candidate artificial receptor array preparation,and detection of lead artificial receptors.

[0320] Synthesis

[0321] Building block synthesis employed a general procedure outlined inScheme 7, which specifically illustrates synthesis of a building blockof general Formula 2 on a tyrosine framework with recognition elementpair A4B4. This general procedure was employed for synthesis of buildingblocks of general Formula 2, with a tyrosine framework and recognitionelement pairs A2B2, A4B4, and A6B6, the structures of which are shown inScheme 8.

[0322] This general procedure was also employed for synthesis ofbuilding blocks of general Formula 2, with a linker, tyrosine framework,and recognition element pairs A4B2 and A4B6, the structures of which areshown in Scheme 9. These two building blocks can be referred to asTyrA4B2 and TyrA4B6, respectively. Building blocks TyrA4B2 and TyrA4B6where readily prepared from the 4-X BOC intermediate by the method ofScheme 10.

[0323] Results

[0324] Synthesis of the desired building blocks proved to be generallystraightforward. These syntheses illustrate the relative simplicity ofpreparing the building blocks with 2 recognition elements havingdifferent structural characteristics or structures (e.g. A4B2, A6B3,etc.) once the building blocks with corresponding recognition elements(e.g. A2B2, A4B4, etc) have been prepared via their X BOC intermediate.

[0325] Synthesis of a building block of general Formula 2 on a tyrosineframework with recognition element pair A4B4 proceeded as illustrated inScheme 7 to yield the quantities of intermediates and product listed onTable 3. TABLE 3 Synthesis Data for Building Block TyrA4B4. Intermediateor Product TARE YIELD A4-X BOC 710 mg 74% YIELD from TYR-BOC 8.20 g 80%(2 synthetic steps) A4B4 ESTER 157 mg 56% YIELD from A4-X BOC 1.26 g 46%(2 synthetic steps) TyrA4B4 75 mg 79% YIELD from A4B4 ESTER 840 mg 88%

[0326] Structures of the two intermediates and of building block TyrA4B4were verified by proton NMR.

[0327] This scheme has also been used to synthesize building blocksTyrA2B2, TyrA4B4, TyrA6B6, TyrA4B2, and TyrA4B6 with the results shownin Table 4: TABLE 4 Synthesis Data for Building Blocks TyrA2B2, TyrA4B2,TyrA4B6, and TyrA6B6. Intermediate or Product TARE YIELD TyrA2B2 A2-XBOC 3.04 g 68% A2B2 ESTER 697 mg 88% TyrA2B2 163 mg 87% TyrA4B2 A4B2ESTER 321 mg 65% TyrA4B2 172 mg 91% TyrA4B6 A4B6 ESTER 173 mg 37%TyrA4B6 75 mg 80% TyrA6B6 A6-X BOC 3.11 g 71% A6B6 ESTER 436 mg 45%TyrA6B6 44 mg 47%

[0328] Summary NMR Data

[0329] NMR conditions were 300 MHz in a solvent mixture ofdeuterochloroform/dmethanol.

[0330]2-X t-BOC R1: -isobutyl R2: t-BOC R3: Linker Ester

[0331] 0.82 (m, 6H, c′, —CH(CH ₃)₂); 1.27 (t, 3H, e″, —O—CH₂—CH ₃); 1.40(s, 9H, t-BOC —C(CH ₃)₃); 1.68 (m, 1H, b′, —CH(CH₃)₂); 2.09 (m, 2H, b″,—C(O)—CH₂—CH ₂—CH₂—O—); 2.52 (t, 2H, c″, —C(O)—CH ₂—CH₂—CH₂—O—);2.82-3.06 (m, 4H, a′ and C _(1,2), ABX); 4.00 (t, 2H, a″,—C(O)—CH₂—CH₂—CH ₂—O—); 4.15 (q, 3H, d″, —O—CH ₂—CH₃); 4.21 (m, 1H, d,ABX); 4.26 (br S, 1H, R1 amide); 6.82 (d, 2H, J=8.6 Hz, a); 7.10 (d, 2H,J=8.6 Hz, b).

[0332] 2-2 R3-ester R1: -isobutyl R2: -cyclopentyl R3: Linker Ester

[0333] 0.82 (m, 6H, c′, —CH(CH ₃)₂); 1.04-1.09 (m, 2H, cyclopentyl);1.27 (t, 3H, e″, —O—CH₂—CH ₃); 1.45-1.75 (m, 10H, B, cyclopentyl, b′,—CH(CH₃)₂); 2.08 (m, 2H, b″, —C(O)—CH₂—CH ₂—CH₂—O—); 2.18 (t, 2H, A);2.52 (t, 2H, c″, —C(O)—CH₂—CH₂—CH ₂—O—); 2.79-3.05 (m, 4H, a′ and C_(1,2), ABX); 3.98 (t, 2H, a″, —C(O)—CH₂—CH₂—CH ₂—O—); 4.15 (q, 3H, d″,—O—CH ₂—CH₃); 4.54 (m, 1H, d, ABX); 4.75 (br S, 2H, R1 and R2 amide);6.81 (d, 2H, J=8.6 Hz, a); 7.12 (d, 2H, =8.8 Hz, b).

[0334] [2-2] R3-COOH R1: -isobutyl R2: -cyclopentyl R3: Linker —COOH

[0335] 0.82 (m, 6H, c′, —CH(CH ₃)₂); 1.03-1.08 (m, 2H, cyclopentyl);1.46-1.73 (m, 10H, B, cyclopentyl, b′, —CH(CH₃)₂); 2.08 (m, 2H, b″,—C(O)—CH₂—CH ₂—CH₂—O—); 2.16 (t, 2H, A); 2.51 (t, 2H, c″, —C(O)—CH₂—CH₂—CH₂—O—); 2.80-3.04 (m, 4H, a′ and C _(1,2), ABX); 3.99 (t, 2H, a″,—C(O)—CH₂—CH₂—CH ₂—O—); 4.54 (m, 1H, d, ABX); 4.68 (br S, 2H, R1 and R2amide); 6.82 (d, 2H, J=8.6 Hz, a); 7.12 (d, 2H, J==8.6 Hz, b).

[0336] 4-2 R3-ester R1: -methoxyphenyl R2: -cyclopentyl R3: Linker Ester

[0337] 1.03-1.08 (m, 2H, cyclopentyl); 1.26 (t, 3H, e″, —O—CH₂—CH ₃);1.45-1.73 (m, 9H, B, cyclopentyl); 2.09 (m, 2H, b″, —C(O)—CH₂—CH₂—CH₂—O—); 2.17 (t, 2H, A); 2.52 (t, 2H, c″, —C(O)—CH ₂—CH₂—CH₂—O—);2.61-2.68 (m, 2H, b′, —CH₂—CH—phenyl); 2.77-3.02 (m, 2H, C _(1,2), ABX);3.26-3.44 (m, 2H, a′, —CH ₂—CH₂-phenyl); 3.78 (s, 3H, phenyl-OCH ₃);3.98 (t, 2H, a″, —C(O)—CH₂—CH₂—CH ₂—O—); 4.14 (q, 3H, d″, —O—CH ₂—CH₃);4.50 (m, 1H, d, ABX); 4.62 (br S, 2H, R1 and R2 amides); 6.81 (d, 2H,a); 6.82 (d. 2H, J=8.8 Hz, d′); 7.04 (d, 2H, J=8.6 Hz, b); 7.08 (d, 2H,J=8.6 Hz, c′).

[0338] [4-2] R3-COOH R1: -methoxyphenyl R2: -cyclopentyl R3: Linker—COOH

[0339] 1.04-1.08 (m, 2H, cyclopentyl); 1.43-1.73 (m, 9H, B,cyclopentyl); 2.08 (m, 2H, b″, —C(O)—CH₂—CH ₂—CH₂—O—); 2.17 (t, 2H, A);2.50 (t, 2H, c″, —C(O)—CH ₂—CH₂—CH₂—O—); 2.62-2.68 (m, 2H, b′, —CH₂—CH₂-phenyl); 2.77-3.02 (m, 2H, C _(1,2), ABX); 3.24-3.44 (m, 2H, a′, —CH₂—CH₂-phenyl); 3.78 (s, 3H, phenyl-OCH ₃); 3.99 (t, 2H, a″,—C(O)—CH₂—CH₂—CH ₂—O—); 4.49 (m, 1H, d, ABX); 6.80-6.85 (m, 4H, a andd′); 7.03-7.09 (m, 4H, b and c′).

[0340] 4-X t-BOC R1: -methoxyphenyl R2: t-BOC R3: Linker Ester

[0341] 1.26 (t, 3H, e″, —O—CH₂—CH ₃); 1.39 (s,9H, t-BOC —C(CH ₃)₃); 2.09(m, 2H, b″, —C(O)—CH₂—CH ₂—CH₂—O—); 2.51 (t, 2H, c″, —C(O)—CH₂—CH₂—CH₂—O—); 2.63-2.67 (m, 2H, b′, —CH₂—CH ₂-phenyl); 2.86-2.93 (m,2H, C _(1,2), ABX); 3.24-3.47 (m, 2H, a′, —CH ₂—CH₂-phenyl); 3.79 (s,3H, phenyl-OCH ₃); 3.98 (t, 2H, a″, —C(O)—CH₂—CH₂—CH ₂—O—); 4.15 (m, 3H,d″, —O—CH ₂—CH₃ and d, ABX); 4.26 (br S, 1H, R1 amide); 6.80-6.83 (m,4H,a and d′); 7.03 (d, 2H, J=8.2 Hz, b); 7.08 (d, 2H, J=8.6 Hz, c′).

[0342] 4-4 R3-ester R1: -methoxyphenyl R2: -cinnamic R3: Linker Ester

[0343] 1.25 (t, 3H, e″, —O—CH₂—CH ₃); 2.08 (m, 2H, b″, —C(O)—CH₂—CH₂—CH₂—O—); 2.51 (t, 2H, c″, —C(O)—CH ₂—CH₂—CH₂—O—); 2.63-2.67 (m, 2H,b′, —CH₂—CH ₂-phenyl); 2.95-3.01 (m, 2H, C _(1,2), ABX); 3.27-3.46 (m,2H, a′, —CH ₂—CH₂-phenyl); 3.73 (s, 3H, phenyl-OCH ₃); 3.97 (t, 2H, a″,—C(O)—CH₂—CH₂—CH2—O—); 4.13 (q, 3H, d″, —O—CH ₂—CH₃); 4.58 (br S, 2H, R1and R2 amides); 4.62 (m, 1H, d, ABX); 6.55 (d, 1H, J=15.9 Hz, A,—CH═CH-phenyl); 6.78-6.83 (m,4H, a and d′); 7.03 (d, 2H, J=8.6 Hz, b);7.12 (d, 2H, J=8.6 Hz, c′); 7.37-7.39 (m, 3H, C,E); 7.52-7.57 (m, 3H, B—CH═CH-phenyl and D).

[0344] [4-4] R3-COOH R1: -methoxyphenyl R2: -cinnamic R3: Linker —COOH

[0345] 2.09 (m, 2H, b″, —C(O)—CH₂—CH ₂—CH₂—O—); 2.50 (t, 2H, c″,—C(O)—CH ₂—CH₂—CH₂—O—); 2.63-2.67 (m, 2H, b′, —CH₂—CH ₂-phenyl);2.95-3.01 (m, 2H, C _(1,2), ABX); 3.32-3.42 (m, 2H, a′, —CH₂—CH₂-phenyl); 3.74 (s, 3H, phenyl-OCH ₃); 3.98 (t, 2H, a″,—C(O)—CH₂—CH₂—CH ₂—O—); 4.46 (br S, 2H, R1 and R2 amides); 4.61 (m, 1H,d, ABX); 6.53 (d, 1H, J=15.9 Hz, A, —CH═CH-phenyl); 6.78-6.83 (m,4H, aand d′); 7.03 (d, 2H, J=8.4 Hz, b); 7.12 (d, 2H, J=8.4 Hz, c′);7.37-7.42 (m, 3H, C,E); 7.51-7.58 (m, 3H, B —CH═CH-phenyl and D).

[0346] 4-6 R3-ester R1: -methoxyphenyl R2: -thioether R3: Linker Ester

[0347] 1.26 (t, 3H, e″, —O—CH₂—CH ₃); 1.97 (s, 3H, B, —S—CH ₃); 2.09 (m,2H, b″, —C(O)—CH₂—CH ₂—CH₂—O—); 2.52 (t, 2H, c″, —C(O)—CH—CH₂—CH₂—O—);2.63-2.69 (m, 2H, b′, —CH₂—CH ₂-phenyl); 2.83-3.06 (m, 2H, C _(1,2),ABX); 3.11 (d, 2H, J=3.7 Hz, A, —C(O)—CH ₂—S—); 3.27-3.48 (m, 2H, a′,—CH ₂—CH₂-phenyl); 3.79 (s, 3H, phenyl-OCH ₃); 3.98 (t, 2H, a″,—C(O)—CH₂—CH₂—CH ₂—O—); 4.14 (q, 3H, d″, —O—CH ₂—CH₃); 4.51 (m, 1H, d,ABX); 4.55 (br S, 2H, R1 and R2 amides); 6.80-6.84 (m, 4H, a and d′);7.05 (d, 2H, J=8.6 Hz, b); 7.10 (d, 2H, J=8.6 Hz, c′).

[0348] [4-6] R3-COOH R1: -methoxyphenyl R2: -thioether R3: Linker —COOH

[0349] 1.96 (s, 3H, B, —S—CH ₃); 2.03-2.10 (m, 2H, b″, —C(O)—CH₂—CH₂—CH₂—O—); 2.50 (t, 2H, c″, —C(O)—CH ₂—CH₂—CH₂—O—); 2.64-2.70 (m, 2H,b′, —CH₂—CH ₂-phenyl); 2.82-3.05 (m, 2H, C _(1,2), ABX); 3.11 (d, 2H,J=4.2 Hz, A, —C(O)—CH ₂—S—); 3.30-3.45 (m, 2H, a′, —CH ₂—CH₂-phenyl);3.79 (s, 3H, phenyl-OCH ₃); 3.99 (t, 2H, a″, —C(O)—CH₂—CH₂—CH ₂—O—);4.51 (m, 1H, d, ABX); 6.81-6.84 (m, 4H, a and d′); 7.05-7.11 (m, 4H, band c′).

[0350] 6-X t-BOC R1: -ether R2: t-BOC R3: Linker Ester

[0351] 1.27 (t, 3H, e″, —O—CH₂—CH ₃); 1.40 (s, 9H, t-BOC —C(CH ₃)₃);2.10 (m, 2H, b″, —C(O)—CH₂CH₂—CH₂—O—); 2.52 (t, 2H, c″, —C(O)—CH₂—CH₂—CH₂—O—); 2.85-3.01 (m, 2H, C _(1,2), ABX); 3.28-3.42 (m, 4H, a′and b′); 3.30 (s, 3H, c′, —OCH ₃); 3.98 (t, 2H, a″, —C(O)—CH₂—CH₂—CH₂—O—); 4.08 (br S, 1H, R1 amide); 4.15 (q, 3H, d″, —O—CH ₂—CH₃); 4.22(m, 1H, d, ABX); 6.82 (d, 2H, J=8.6 Hz, a); 7.10 (d, 2H, J=8.6 Hz, b).

[0352] 6-6 R3-ester R1: -ether R2: -thioether R3: Linker Ester

[0353] 1.27 (t, 3H, e″, —O—CH₂—CH ₃); 1.99 (s, 3H, B, —S—CH ₃); 2.09 (m,2H, b″, —C(O)—CH₂—CH ₂—CH₂—O—); 2.52 (t, 2H, c″, —C(O)—CH ₂—CH₂—CH₂—O—);2.87-3.10 (m, 2H, C _(1,2), ABX); 3.13 (d, 2H, J=4.6 Hz, A, —C(O)—CH₂—S—); 3.29-3.44 (m, 4H, a′ and b′); 3.33 (s, 3H, c′, —OCH ₃); 3.99 (t,2H, a″, —C(O)—CH₂—CH₂—CH ₂—O—); 4.15 (q, 3H, d″, —O—CH ₂—CH₃); 4.55-4.60(m, 1H, d, ABX); 4.57 (br S, 2H, R1 and R2 amide); 6.82 (d, 2H, J=8.6Hz, a); 7.13 (d, 2H, J=8.6 Hz, b).

[0354] [6-6] R3-COOH R1: -ether R2: -thioether R3: Linker —COOH

[0355] 1.98 (s, 3H, B, —S—CH ₃); 2.08 (m, 2H, b″, —C(O)—CH₂—CH₂—CH₂—O—); 2.51 (t, 2H, c″, —C(O)—CH ₂—CH₂—CH₂—O—); 2.86-3.10 (m, 2H, C_(1,2), ABX); 3.13 (d, 2H, J=4.8 Hz, A, —C(O)—CH ₂—S—); 3.28-3.44 (m,4H, a′ and b′); 3.33 (s, 3H, c′, —OCH ₃); 3.99 (t, 2H, a″,—C(O)—CH₂—CH₂—CH ₂—O—); 4.55-4.60 (m, 1H, d, ABX); 6.83 (d, 2H, J=8.8Hz, a); 7.14 (d, 2H, J=8.4 Hz, b).

Example 2 Preparation of Candidate Artificial Receptors

[0356] The comparatively small numbers of candidate artificial receptorsmade from combinations of 3 or 5 building blocks were prepared in 12×75borosilicate glass test tubes. The inner surface of the tube wasmodified using standard glass derivatization chemistry. The modifiedtubes were convenient vessels for the test ligand binding experiments.

[0357] The three tyrosine framework building blocks TyrA2B2, TyrA4B4,and TyrA6B6 resulted in three receptors homogeneous in building block(A2B2, A4B4, and A6B6), three receptors heterogeneous in building blockand containing two building blocks (A2B2 plus A4B4, A2B2 plus A6B6, andA4B4 plus A6B6), and one receptor heterogeneous in building block andcontaining three building blocks (A2B2 plus A4B4 plus A6B6).

[0358] Preparation of Amino-Glass

[0359] The first step in the tube or other glass derivatization processwas to covalently immobilize a pendant functional group on the glasssurface. The reaction of an aminoethyl silicon reagent with the glasswas a straightforward method for the introduction of a pendant aminewhich was subsequently used for receptor preparation (FIG. 5). Aminemodification was accomplished by the protocol of Schreiber (MacBeath etal. (1999) J. Am. Chem. Soc., 121, 7967-7968).

[0360] Briefly, tubes were soaked in water for 1-2 hr then drained for30-60 min. The tubes are then treated overnight with “Piranha” solution:70/30 (v/v) conc. H₂SO₄/30% H₂O₂. Each 12×75 received about 0.6 mL ofthis solution and was loosely covered with aluminum foil. The next day,the solution was decanted and the tubes were rinsed with water anddrained. The tubes were then treated with amino silane solution. Thetubes were filled with 0.5 mL of a solution of 3% amino silane in 95%ethanol, covered with foil, and allowed to stand for 60 min. Thesolution was decanted. The tubes were rinsed with ethanol, drained, andthen heated at 125° C. for 60 min. The tubes were rinsed again withethanol, drained, and allowed to dry overnight.

[0361] Evaluation of Amino-Glass

[0362] The load of amine on the glass surface was determined by themethod provided by Pierce Chemical Co. as modified by Schreiber(MacBeath et al. (1999) supra). The method was further modified toinclude THF/H₂O washes to remove non-covalently bound label. Thismethod, including the additional step, gave consistent semi-quantitativeresults.

[0363] Briefly, the method employed in these studies included adding tothe amino tubes about 2 ml of pH 8.5 NaHCO₃ (4.20 grams of NaHCO₃ perliter) and soaking for 5 min. The bicarbonate solution was decanted andthe tubes drained. The tubes were then reacted with an SDTB (Pierce)labeling solution (SDTB in HPLC grade DMF and pH 8.5 NaHCO₃). Thesolution was made and immediately added to the tubes, which then wereshaken for 30-45 min. The solution was decanted and the tubes werewashed repeatedly with water and THF and then water. After addition of 1mL of 30% perchloric acid, OD at 498 nm was read. A load of 2 amines persquare nm gives an OD of 0.07 for 12×75 mm tubes.

[0364] Over several batches of tubes (about 1000 tubes), loading ofabout 2.3 amines per square nm was achieved. Such amine loads were wellwithin the densities required for these studies.

[0365] Preparation of Candidate Artificial Receptors

[0366] Functionalization of the amine modified glass surface wasaccomplished by reaction with activated carboxyl derivatives to form theamide (see, e.g., FIG. 5). This reaction employed the linker carboxyl incertain embodiments of the building blocks, e.g., certain embodimentshaving Formula 2.

[0367] In the present example, coupling of linker carboxyl containingbuilding blocks to the amine support matrix was conducted generallyaccording to established methods for coupling carboxyl containingcompounds to amines on supports (the Pierce method (Pierce Chemical Co.)as described by Schreiber (MacBeath et al. (1999) supra)). The buildingblock linker carboxyl group was activated by reacting the building blockwith carbodiimide in the presence of sulfo N-hydroxysuccinimide in aq.DMF solution. After overnight activation of the carboxyl to the sulfoNHSactivated ester, the building block was reacted directly with the aminoglass. Coupling combinations of building blocks to the amino-glass wasaccomplished by premixing of activated building blocks prior to additionto the amino tube. Support matrix amines not reacted with building blockwere acetylated by the same general method.

[0368] Briefly, building block amide and other glass supports wereprepared by adding to amino-glass tubes 2 mL of pH 8.5 NaHCO₃ for 10min. The tubes were decanted and drained. Activated building block(s) oracetic anhydride were dissolved in DMF/pH 8.5 NaHCO₃ and added to theamino-glass tubes. The tubes were shaken for 60 min, decanted, andwashed with aq. THF and/or water. The tubes were used immediately andalso after drying and storage. Activated carboxyl groups were typicallyin more than 50-fold excess over amines.

[0369] Derivatized tubes prepared by this procedure included thoselisted in Table 5. TABLE 5 Summary of Amide Tubes Prepared. TUBEDESCRIPTION —NH2 pendant amine —Ac acetamide −22 Homogeneous immobilizedbuilding block TyrA2B2 −44 Homogeneous immobilized building blockTyrA4B4 −66 Homogeneous immobilized building block TyrA6B6 −22/44Candidate artificial receptor TyrA2B2 plus TyrA4B4 (building blockheterogeneous) −22/66 Candidate artificial receptor TyrA2B2 plus TyrA6B6(building block heterogeneous) −44/66 Candidate artificial receptorTyrA4B4 plus TyrA6B6 (building block heterogeneous) −22/44/66 Candidateartificial receptor TyrA2B2, TyrA4B4, plus TyrA6B6 (building blockheterogeneous)

[0370] Building block incorporation was determined as described abovefor evaluation of amino-glass. This evaluation indicated that the amideforming reaction produced candidate artificial receptors includingsubstantial amounts of building block, for example, 30 to 80% of theamines were derivatized by building block. As shown below, binding tothe candidate artificial receptor was observed when 30% or more ofamines were derivatized with building block.

Example 3 Screening Test Ligands Against Candidate artificial receptorsMade From 3 Building Blocks

[0371] In this example, candidate artificial receptors were tested fortheir ability to bind to test ligands. The test ligands were coupledhorseradish peroxidase (HRP). Conjugates of test ligand with HRP werereadily prepared by known methods, were stable in solution, and weredetected in picogram quantities.

[0372] Materials and Methods

[0373] Preparation of Labeled Test Ligand

[0374] Conjugates of HRP and test ligand were prepared by firstmodifying HRP to incorporate additional pendant amine groups. Briefly,EIA grade HRP (e.g., SIGMA P6782) was dissolved in water and oxidizedwith NaIO4 at about 4° C. in the dark or in subdued light. The oxidizedHRP was subjected to gel filtration chromatography (e.g., SEPHADEX® G-25equilibrated with 100 mM pH 9.4 borate buffer). The resulting solutionof oxidized HRP was reacted with ethylene diamine dihydrochloride forabout 30 min. at 4° C. The derivatized HRP was then reduced with NaBH₄to yield amine derivatized HRP (amino-HRP). The amino-HRP was thendialyzed against the borate buffer for about 8 hours with a singlechange of dialysis solution.

[0375] Then, the amino-HRP was further modified to form amide links tothe test ligand. Briefly, a carboxyl group containing derivative of thetest ligand was converted to an activated ester using the methoddescribed above for building blocks. The activated test ligand and theamino-HRP were reacted overnight with eventual addition of 10-100 foldexcess of activated test ligand to amines on the amino-HRP. Theconjugate of test ligand with HRP(HRP-ligand conjugate) was purified bygel filtration chromatography and/or dialysis, as described above. Theseconjugates were stored at 4° C. in PBS solution with 20 μl Tween-20added per liter of PBS. Analyte load was determined by the UV/Visabsorbance of the analyte and/or by amine loss.

[0376]FIG. 14 illustrates HRP (Formula H1), HRP derivatives (Formulas H2and H3), and conjugates of test ligand and HRP (Formulas H4, H5, and H6)that have been made for and used in these examples. HRP has a molecularweight of 40,000 and 2 free amines in its native form. Native HRP wasoxidized to form amine HRP with about 20 amino groups on its surface.Amide derivatives of amino HRP were formed by reacting the amino HRPwith a anhydride or acid chloride. Preferred amide HRPs include theacetamide derivative. HRP test ligand conjugates were formed, forexample, by reacting amine HRP with an activated ester form of a testligand.

[0377] Evaluating Binding of Test Ligands to Candidate ArtificialReceptors

[0378] Candidate artificial receptors were prepared as described inExample 2. Binding of test ligand to candidate artificial receptors wasevaluated by the following procedure. Briefly, one or more tubes, eachcontaining a candidate artificial receptor, were rinsed with PBS,decanted, and drained. PBS (250 μl) was added to the tube, HRP-ligandconjugate was added (20 μl), and the candidate artificial receptors wereincubated at room temperature for the desired time, for example, 30 minor longer. The 20 μl aliquot of HRP conjugate typically included aconcentration of 1.0 μg/ml, 0.1 μg/ml, and/or 0.01 μg/ml of theconjugate. This concentration in the aliquot is referred to as the testconcentration and is shown on Figures. The tubes were decanted, rinsedtwice with PBS, rinsed with water, decanted, and drained. Then color wasdeveloped with an HRP substrate, for example, the HRP chromogen (source:BioFX Corp.). Typically, 450 μl of substrate was added and the tubeswere incubated for 15 min. Then, the chromogen solution was quicklytransferred to a clean test tube and 600 μl of stop solution (0.1 N HCl)was added. The stopped tubes were read at 450 nm.

[0379] The receptor tubes, which were not exposed to the strongly acidicstop solution, were prepared for reuse in subsequent experiments byrinsing with water and with PBS, followed by addition of 2 ml PBS. Thetubes were soaked with buffer and rinsed as needed to remove the boundHRP-ligand conjugate.

[0380] Results and Discussion

[0381] Experiment 1:

[0382] Experiment 1 demonstrated at least that:

[0383] a) the relative binding of a particular HRP-ligand conjugate wasconsistently reproduced over a series of tube preparations and over aperiod of several weeks;

[0384] b) that HRP-ligand conjugates gave differential responses tosimple floor/receptor surfaces;

[0385] c) the nature of the floor played a role in binding.

[0386] The differential response to simple floor/receptor surfaces isillustrated by the data presented in Table 6. The data in the last tworows of this table demonstrate that the nature of the group derivatizingremaining amines (those not reacted with building block) affectedbinding to a candidate receptor. In this experiment, the test ligandbound better to the building block with free amine on the “floor”compared to building block with acetamide floor. TABLE 6 Test LigandBinds Better to an Immobilized Building Block than to Floor SurfacesTest Ligand, 1.0 μg/ml (as ligand-HRP conjugate) Tube or Building BlockOD (std dev) Acetate bare amino tube  0.14 (0.03) Acetate acetylatedamino tube  0.05 (0.03) Acetate TyrA4B4 >2.8 TCDD bare amino tube  1.65(0.39) TCDD acetylated amino tube  0.11 (0.09) TCDD TyrA4B4 >2.8 TCDDTyrA4B4 with acetylated  0.77 floor

[0387] Experiment 2:

[0388] Experiment 2 demonstrated at least that:

[0389] a) Receptor binding was sensitive to both the structure and theconcentration of the HRP derivative.

[0390] b) The HRP moiety was not a significant factor in the observedbinding patterns. Binding of the HRP—NH—Ac control was minimal withrespect to test ligand binding

[0391] c) Binding was controlled by both kinetic and thermodynamicfactors.

[0392] d) Simple partition coefficient driven equilibria were notresponsible for the observed test ligand binding to the inventivecandidate artificial receptors.

[0393] e) An unknown sample containing a test ligand can be identifiedby its distinct binding pattern.

[0394] In Experiment 2, six test ligands (as ligand-HRP conjugates) weretested against 4 candidate artificial receptors, 3 homogenousimmobilized building blocks, acetylated amino-glass, and amino-glass.The results of Experiment 2 are illustrated in FIGS. 15-18.

[0395]FIG. 15 illustrates bar charts of the binding pattern comparisonfor native HRP, acetylated-HRP, and the TCDD derivative of amino-HRP.The values in FIG. 15 were taken from the mean values listed in Table 6.This Figure illustrates binding of this test ligand and these controlHRP derivatives to amino-glass, to acetylated amino-glass, to each ofhomogeneous immobilized building blocks TyrA2B2, TyrA4B4, and TyrA6B6,and to candidate artificial receptors TyrA2B2 plus TyrA4B4; TyrA2B2 plusTyrA6B6; TyrA4B4 plus TyrA6B6; and TyrA2B2, TyrA4B4, plus TyrA6B6. TheHRP derivatives were tested at 1 μg/ml (20 μL of which includes only 20ng of HRP, or picomole amounts of the test ligand) against the suite of9 control, building block, and receptor surfaces. In this experiment ODvalues were linear up to about 2.4 and then increased non-linearly.

[0396] Note that these data are consistent with the results ofExperiment 1 and extend those first results to demonstrate that receptorbinding was sensitive to both the structure and the concentration of theHRP derivative. The results illustrated in FIG. 15, which include thebinding pattern for native HRP, also demonstrate that the HRP moiety wasnot a significant factor in the observed binding patterns when comparedto the binding of a ligand-HRP conjugate, e.g. HRP—NH-34K, HRP—NH-ETU,or HRP—NH-TCDD.

[0397] Binding of the HRP moiety, which was used as the binding screenlabel, to candidate receptors would cause false positives. FIG. 15illustrates that native HRP did not show significant non-specificbinding to either the control or candidate receptor tubes at the highestconcentration of HRP used for these studies.

[0398]FIG. 16 illustrates bar charts showing the reproducibility of thebinding pattern for amino-H RP to amino-glass, to acetylatedamino-glass, to each of homogeneous immobilized building blocks TyrA2B2,TyrA4B4, and TyrA6B6, and to candidate artificial receptors TyrA2B2 plusTyrA4B4; TyrA2B2 plus TyrA6B6; TyrA4B4 plus TyrA6B6; and TyrA2B2,TyrA4B4, plus TyrA6B6. FIG. 16 illustrates that both the relativebinding OD and binding pattern were consistent over a triplicate screenof HRP—NH₂ versus the 9 control, building block, and receptor surfaces.The data illustrated in FIG. 16 show that pattern of relative OD valuesfor each homogeneous immobilized building block or receptor wasessentially the same between the trials. The following order wasobserved from highest to lowest OD: 1) TyrA4B4; 2-4) amino glassTyrA2B2, and TyrA2B2 plus TyrA4B4; 5-6) TyrA4B4 plus TyrA6B6, andTyrA2B2, TyrA4B4 plus TyrA6B6; 7-9) acetylated amino glass, TyrA6B6, andTyrA2B2 plus TyrA6B6.

[0399]FIG. 17 illustrates bar charts of the binding pattern comparisonfor native HRP, amino-HRP, the 34K derivative of amino-HRP (Formula H4),the TCDD derivative of amino-HRP (Formula H6), and the ETU derivative ofamino-HRP (Formula H5). These were tested at 1 μg/ml, 0.1 μg/ml, and0.01 μg/ml. This Figure illustrates binding of this test ligand andthese control derivatives to amino-glass, to acetylated amino-glass, toeach of homogeneous immobilized building blocks TyrA2B2, TyrA4B4, andTyrA6B6, and to candidate artificial receptors TyrA2B2 plus TyrA4B4;TyrA2B2 plus TyrA6B6; TyrA4B4 plus TyrA6B6; and TyrA2B2, TyrA4B4, plusTyrA6B6. The results shown in FIG. 17 demonstrate observed concentrationdependent binding, which reflected the different binding affinities ofthe receptor surfaces for the test ligands and differential patterns ofbinding to the control, building block, and receptor surfaces by thetest ligands.

[0400] The target screen was based on the binding of HRP labeled testligand. Binding of the HRP—NH-Ac derivative should be minimal withrespect to binding of HRP-test ligand conjugate to give the best signalfor target binding. As illustrated by the data for HRP—NH—Ac (0.1×concentration) compared to the HRP—NH-34K, ETU, TCDD conjugates (FIG.17), the binding of the HRP—NH-Ac control was minimal with respect totest ligand binding.

[0401]FIG. 18 illustrates bar charts of the binding pattern comparisonfor the ETU derivative of amino-HRP (Formula H5) using two differentprotocols for determining binding. In the kinetic protocol, the HRPconjugate at 0.1 and 0.01 μg/ml was added to the tube, incubated, thendecanted. The tubes were rinsed and HRP chromogen was developed withinabout 30 min of adding ligand to receptor. In the thermodynamicprotocol, HRP conjugate at 1 μg/ml and was added to the tube anddecanted after incubation. Then the tube was rinsed with buffer, morebuffer was added to the tube, and it was incubated overnight (overnight#1). This rinse, adding, and incubating was repeated for overnight #2.This Figure illustrates binding of this test ligand and these controlderivatives to amino-glass, to acetylated amino-glass, to each ofhomogeneous immobilized building blocks TyrA2B2, TyrA4B4, and TyrA6B6,and to candidate artificial receptors TyrA2B2 plus TyrA4B4; TyrA2B2 plusTyrA6B6; TyrA4B4 plus TyrA6B6; and TyrA2B2, TyrA4B4, plus TyrA6B6.

[0402] The results shown in FIG. 18 demonstrate that binding wascontrolled by both kinetic and thermodynamic factors. This extends theresults discussed above. The kinetic assay detects those candidateartificial receptors to which the test ligand binds quickly, kineticfactors predominate. The thermodynamic assay detects those candidateartificial receptors to which the test ligand binds more slowly but moretightly, thermodynamic factors predominate. The pattern of binding tothe 9 control, building block, and receptor surfaces for the kineticprotocol (addition of HRP and 30 minute incubation) was different fromthe thermodynamic protocol (addition of 1.0×HRP and 30 minute incubationfollowed by an overnight incubation in buffer), but the patterns wereconsistent within each series.

[0403]FIG. 19 illustrates bar charts of the binding pattern comparisonfor the ETU derivative of amino-HRP (Formula H5) using protocols similarto those used in the experiments of FIG. 18. In the present experiment,the tubes were incubated for varying times before the buffer solutionwas decanted. FIG. 19 illustrates that both the extent of binding, asmeasured by OD value, and the pattern of binding changed as the HRP isincubated for increasing periods of time. These results reflect akinetic response at early times and then a thermodynamic equilibriumresponse at later times.

[0404] Binding Evaluation: The Hydrophobic/Lipophilic Component.

[0405] Hydrophobic/lipophilic interactions can play a potentiallysignificant role in the binding of a test ligand to a candidatereceptor. However, it is relevant to demonstrate that simple partitioncoefficient driven equilibria were not responsible for the observed testligand binding to the inventive candidate artificial receptors. Inaddition, it is relevant to define the role that simplelipophilic/hydrophobic partitioning plays in the present Examples. Infact, the present binding results were not simply the result oflipophilic/hydrophobic interactions.

[0406] Test Ligand LogP Versus Binding

[0407] The LogP values for the —NH₂, —NH—Ac, —NH-34K, —NH-ETU and—NH-TCDD test ligands which were used for this study were calculatedusing the ACD/LogD Suite program (Advanced Chemistry Development Inc.,Toronto, Canada). The values obtained were: TARGET LogP —NH2 −1.74—NH—Ac −1.05 —NH-ETU +0.26 —NH-34K +2.28 —NH-TCDD +3.84

[0408]FIG. 20 presents the 3 bar graphs (based on data presented in FIG.16) along with LogP data. Pairwise comparison of individual resultsindicate that simple partitioning does not explain the observed results.For example, the binding (OD) values for HRP—NH-34K, -ETU and -TCDD onthe homogeneous, single building block TyrA4B4 tubes spanned a rangewhich was less than a factor of two while LogP spanned several orders ofmagnitude.

[0409] The binding (OD) data from the study with combinations of threebuilding blocks in candidate receptors has been reorganized in Tables 7and 8 to include LogP. FIG. 21 plots the OD data for the 0.1 μg/mlHRP-test ligand conjugate (Table 8) versus LogP. The upper graph in FIG.21 plots the values for the n=1, homogeneous building blocks. The lowergraph plots the values for candidate receptors.

[0410] In the upper graph in FIG. 21, the plots for the alkanesubstituted building block TyrA2B2 and the phenyl substituted buildingblock TyrA4B4 generally conform to the expectation that bindingincreased with increasing target lipophilicity as indicated byincreasing LogP. The plot for the hydrophilic building block TyrA6B6also generally conforms to expectations as binding was strongest for themore polar/hydrophilic ETU test ligand when compared to binding of themore lipophilic 34K and TCDD test ligands. The results for 1.0 μg/mlHRP-test ligand were generally consistent (Table 7) and support theconclusion that lipophilic interactions play a role in target bindingfor the higher LogP targets. TABLE 7 Binding (OD) data from combinationsof three building blocks in candidate receptors including LogP. —NH-—NH- —NH- HRP-NH2 —NH—Ac ETU 34K TCDD LOG P -> TUBE −1.74 −1.05 +0.26+2.28 +3.84 1. —NH2 0.77 0.19 >2.8 2.45 2.67 2. —NH—Ac 0.11 0.06 >2.81.52 0.34 3. -22 0.69 1.14 >2.8 >2.8 >2.8 4. -44 1.41 0.56 >2.8 >2.82.75 5. -66 0.17 0.04 2.78 0.68 0.12 6. -22/44 0.66 2.06 >2.8 2.15 2.187. -22/66 0.14 0.23 >2.8 >2.8 1.39 8. -44/66 0.36 0.99 >2.8 >2.8 2.38 9.-22/44/66 0.28 1.68 >2.8 2.59 1.31

[0411] TABLE 8 Binding (OD) data from combinations of three buildingblocks in candidate receptors including LogP. —NH- —NH- —NH- HRP-NH2—NH—Ac ETU 34K TCDD LOG P -> TUBE −1.74 −1.05 +0.26 +2.28 +3.84 1. —NH2<0.04 <0.04 0.86 0.05 0.22 2. —NH—Ac <0.04 <0.04 0.20 0.05 0.08 3. -22<0.04 <0.04 0.80 0.86 0.51 4. -44 <0.04 <0.04 1.34 1.25 2.19 5. -66<0.04 <0.04 0.68 0.17 0.04 6. -22/44 <0.04 <0.04 1.00 0.90 0.27 7.-22/66 <0.04 <0.04 1.02 0.42 0.44 8. -44/66 <0.04 <0.04 1.10 0.93 0.879. -22/44/66 <0.04 <0.04 0.55 0.51 0.50

[0412] The plots obtained for the combinations of two and three buildingblocks in candidate receptors provide an extended perspective on therole of lipophilic interaction (FIG. 21, lower graph). The conclusionfrom these plots is that although lipophilic driven partitioning mayhave played a role, it was not the dominant factor. For example, theplot for the candidate receptor made from the combination of TyrA2B2plus TyrA4B4, which was the most lipophilic combination of buildingblocks, significantly decreased as LogP increased above the ETU value of0.26. The OD values for ETU, 34K and TCDD binding to the candidatereceptor made from the combination of TyrA4B4 plus TyrA6B6 were similar(1.10, 0.93 and 0.87 OD respectively) even though their LogP values spanmore than 3 orders of magnitude (0.26 to 3.84). Clearly, lipophilicbinding was not the major factor in test ligand binding by thesecandidate receptors.

[0413] Binding Evaluation: Test Ligand Binding Patterns

[0414] Binding Pattern Interpretation

[0415]FIG. 22 presents the data from this example for the candidatereceptors with combinations of 2 and 3 building blocks versus the Accontrol target and the three 34K, TCDD and ETU test ligands. It is clearfrom FIG. 22 that the test ligands bound to the candidate receptors withdifferent binding patterns. An unknown sample that contained one ofthese four HRP conjugates could be readily identified by its distinctpattern.

[0416] Binding Affinity

[0417] It is significant to note that the observed binding affinities,even after testing a suite of only 4 candidate artificial receptors and3 building block surfaces, spanned several orders of magnitude (FIG.17). An estimate of binding affinity for the best receptor (TyrA2B2 plusTyrA4B4) for the ETU conjugate gives a range of K_(Binding) of 2×10⁴ to6×10⁵ L/M.

[0418] Reproducibility

[0419] Binding of ETU to the suite of 9 control, building block, andreceptor surfaces gave OD readings from replicate experiments that werereproducible to within 5-20% (CV) for the different tubes. Certain ofthe tubes used in these experiments have produced good results throughrepeated use over a period of several months.

[0420] Conclusions

[0421] Identification of an optimum (specific, sensitive) workingartificial receptor from the limited pool of 9 control, building block,and receptor surfaces was not expected and not likely. Rather, the goalof these two experiments was to demonstrate that candidate artificialreceptors could be assembled and tested to provide one or more leadartificial receptors. This has been successfully demonstrated. Plus, aworking artificial receptor complex was identified.

Example 4 Screening Test Ligands Against Candidate artificial receptorsMade From 5 Building Blocks

[0422] In this example, test ligands were evaluated against a broaderrange of candidate artificial receptors including combinations of up to4 building blocks and made from a total of 5 building blocks.

[0423] Materials and Methods

[0424] Building Blocks

[0425] Building blocks were made as described in Example 1.

[0426] Candidate Artificial Receptors

[0427] Tubes with modified amino groups, homogeneous immobilizedbuilding blocks, and candidate artificial receptors includingcombinations of 2, 3 and 4 building blocks were prepared as described inExample 2. This resulted in a set of 34 control, building block, andreceptor tubes (Table 9). The tubes with modified amino groups aredesignated as Floor tubes in Table 9. The tubes with homogeneousimmobilized building blocks are designated as n=1 (number of buildingblocks immobilized in tube equals 1) tubes in Table 9. The tubes withcandidate artificial receptors are designated as n=2, n=3, and n=4 tubesin Table 9. Table 9 lists the order in which results for the floortubes, immobilized building blocks, candidate receptors withcombinations of 2 building blocks, candidate receptors with combinationsof 3 building blocks, and candidate receptors with combinations of 4building blocks appear in FIGS. 23 and 24. TABLE 9 Identification gridfor artificial receptors made from a set of 5 building blocks incombinations of 2, 3, and 4. Results are shown in FIGS. 23 and 24. FLOORf1. —NH2 f2. —Ac[—NH—C(O)—CH3] f3. —SA[—NH—C(O)—CH₂CH₂—COOH] f4.-phenyl[—NH—C(O)-phenyl] Immobilized Building Blocks n1.1 −22 n1.2 −42n1.3 −44 n1.4 −46 n1.5 −66 Candidate Receptors with Combinations of 2Building Blocks n2.1 −22/42 n2.2 −22/44 n2.3 −22/46 n2.4 −22/66 n2.5−42/44 n2.6 −42/46 n2.7 −42/66 n2.8 −44/46 n2.9 −44/46 n2.10 −46/66Candidate Receptors with Combinations of 3 Building Blocks n3.1−22/42/44 n3.2 −22/42/46 n3.3 −22/42/66 n3.4 −22/44/46 n3.5 −22/44/66n3.6 −22/46/66 n3.7 −42/44/46 n3.8 −42/44/66 n3.9 −42/46/66 n3.10−44/46/66 Candidate Receptors with Combinations of 4 Building Blocksn4.1 −22/42/44/46 n4.2 −22/42/44/66 n4.3 −22/42/46/66 n4.4 −22/44/46/66n4.5 −42/44/46/66

[0428] Results and Discussion

[0429] Example 4 demonstrated at least that:

[0430] a) Test ligands displayed distinctive binding to a larger groupof artificial receptors.

[0431] b) Various features within the receptor site cooperate to producetest ligand binding which is greater than the sum of the individualinteractions.

[0432] The set of 34 tubes was screened versus several of the ligand-HRPconjugates and control HRP derivatives. FIGS. 23 and 24 illustrate thatthe control HRP derivative (0.1 μg/ml acetylated amino-HRP) exhibitedminimal binding with this expanded set of candidate receptors, while theligand-HRP conjugate 34K-HRP at 0.1 μg/ml displayed distinctive binding.

[0433] The target screen was based on the binding of HRP labeled testligand. The results of Example 3 demonstrated that the binding of theHRP—NH—Ac control was minimal with respect to test ligand conjugatebinding. This conclusion is further substantiated by comparing the morecomprehensive data set from the N=5, 34 tube experiments. FIGS. 23 and24 demonstrate the minimal binding of HRP—NH—Ac (0.1×) when compared toHRPNH-34K (0.1×). For example, only two tubes showed an OD of greaterthan 0.2 for the HRP—NH—Ac. Considering the 32 tubes for HRP—NH—Ac thatshowed OD<0.2, the mean OD was 0.05 with a standard deviation of 0.04.The data in FIG. 24 show 5 tubes that had OD greater than 0.5. The 27tubes with OD<0.5 showed a mean OD of 0.28 with a standard deviation of0.12.

[0434] Binding Evaluation: The Hydrophobic/lipophilic Component,Continued Comparison to the Lipophilic Mean

[0435] The data from this example for HRP—NH-34K also providesinformation on the role of lipophilic interactions in the observedbinding (FIG. 24). For example, if it is assumed, for the sake of anhypothesis, that the binding OD observed for the single building block,homogeneous/lipophilic building block tubes is predominantly a result oflipophilic partitioning (note that aromatic recognition elements canalso exhibit pi bonding, etc.), then the mean binding observed for the[f-phenyl], TyrA2B2, TyrA4B2, and TyrA4B4 tubes (each of which includesbuilding blocks with similar recognition elements) should be equivalentto the ‘lipophilic component’. The mean for these four tubes was: mean0.40 OD, StdDev 0.15. Clearly, examination of the binding data in FIG.24 indicate that the lipophilic component of binding was not the onlyfactor which contributes to the observed binding. For example, thecandidate receptor made from the combination of TyrA4B2 plus TyrA4B4produced an OD 1.51 and the candidate receptor made from the combinationof TyrA2B2, TyrA4B2, TyrA4B4, plus TyrA4B6 produced OD 0.82. Thesereceptors include lipophilic/hydrophobic recognition elements likeTyrA4B2 and TyrA4B4, but produced greater binding than the lipophilicmean.

[0436] Binding Evaluation: Test Ligand Binding Patterns, Continued

[0437] Demonstration of Recognition Element Cooperative Binding

[0438] An essential feature of selective and sensitive binding by areceptor is that the various binding elements within the receptor sitecooperate to produce test ligand binding which is greater than the sumof the individual interactions. Table 10 compares expected binding (OD),if binding was simply an average effect produced by the interactions ofthe separate building blocks, with the observed binding (OD) for severalof the more prominent values illustrated in FIG. 24. The premise of thiscomparison is that binding could be simply the result of the average ofthe interactions of the separate building blocks if a simplepartitioning mechanism is dominant. Alternatively, binding is morelikely to be a cooperative sum of the individual interactions. The datain Table 10 demonstrate that there was a significant (2 to 4-fold)enhancement of binding for the heterogeneous candidate receptorsincluding combinations of 2, 3 or 4 building blocks when compared totheir homogeneous counterparts including only a single building block.The data was from HRPNH-34K versus the set of building blocks (FIG. 24).The Component Average (Expected) value was calculated from the observedOD for the appropriate single building block components, e.g. forTyrA2B2 plus TyrA4B2 the component expected was the average of 0.63 ODfor TyrA2B2 and 0.32 OD for TyrA4B2 which was 0.48 OD. TABLE 10Comparison of average versus observed binding. Building Blocks WithSimilar Recognition Elements (n = 1) ID OD n1.1 TyrA2B2 0.63 n1.2TyrA4B2 0.32 n1.3 TyrA4B4 0.42 n1.4 TyrA4B6 0.40 n1.5 TyrA6B6 0.06Selected Building Block Distinct Recognition Elements (n = 2, 3, 4)COMPONENT RECEPTOR OBSERVED AVERAGE OBSERVED/ ID OD (EXPECTED) EXPECTEDn2.1 0.42 0.48 0.88 TyrA2B2 plus TyrA4B2 n2.5 1.51 0.37 4.1 TyrA4B2 plusTyrA4B4 n2.7 0.56 0.19 2.9 TyrA4B2 plus TyrA6B6 n2.10 0.51 0.23 2.2TyrA4B6 plus TyrA6B6 n3.8 0.56 0.27 2.1 TyrA4B2 plus TyrA4B4/66 n4.10.82 0.44 1.9 TyrA2B2, TyrA4B2, TyrA4B4, plus TyrA4B6

[0439] Heterogeneous Binding Elements: Significance

[0440] The building blocks had two recognition elements. Building blocksthat had recognition elements which are similar in structure andproperties, e.g. building blocks TyrA2B2, TyrA4B4 and TyrA6B6, aredescribed as having similar recognition elements. Building blocks whichthat had recognition elements with structures that are different instructure and properties, e.g. building blocks TyrA4B2 and TyrA4B6, aredescribed as having distinct recognition elements. The binding patternshown in FIG. 24 has 6 peaks which indicate binding was above the meanfor the data set. Table 11 lists the building block composition of thesecandidate receptors TABLE 11 The building block composition of candidatereceptors of FIG. 24 BUILDING BLOCKS TUBE 2-2 4-2 4-4 4-6 6-6 n2.1 2-24-2 n2.5 4-2 4-4 n2.7 4-2 6-6 n2.10 4-6 6-6 n3.8 4-2 4-4 6-6 n4.1 2-24-2 4-4 4-6 OCCURRENCE RATIO 2/6 5/6 3/6 2/6 3/6

[0441] Clearly, the 4-2 Building Block played a significant role in thebinding of the HRP—NH-34K test ligand. This observation confirms thatthe building blocks which were prepared from heterogeneous recognitionelements played a key role in artificial receptor development.

[0442] Conclusions

[0443] These results demonstrate that there was a significantenhancement of binding for the heterogeneous (n=2,3,4) candidatereceptors when compared to their homogeneous (n=1) counterparts. Whencombined with binding pattern recognition and the demonstratedimportance of both heterogeneous recognition elements and heterogeneousbuilding blocks, these results clearly demonstrate that the presentartificial receptors performed and will perform as expected to achievethe goal of target specific and sensitive artificial receptordevelopment.

Example 5 Preparation of Microarrays of Candidate Artificial Receptors

[0444] Ultimately, the candidate artificial receptors will be presentedin a microarray format on, for example, glass slides. The preparation ofmicroarrays will employ known procedures for evaluation and optimizationof robotic plate preparation and microarray high throughput screeningsystems. Studies with microarrays will extend the current results toevaluation of an array made from 10 building blocks, an array made from18 building blocks, and an array made from 81 building blocks.Microarrays will be made from a 10 building block set including TyrA2B2,TyrA2B4, TyrA4B2, TyrA4B4, TyrA4B6, TyrA6B4, TyrA6B6, TyrA6B8, TyrA8B6,and TyrA8B8. A set of 10 building blocks will be combined to provide 10spots of homogeneous immobilized building block, 45 spots of candidateartificial receptors with two building blocks, 120 spots of candidateartificial receptors with three building blocks, and 210 spots ofcandidate artificial receptors with four building blocks. Microarrayswill be made from an 18 building block set including TyrA2B2, TyrA2B4,TyrA4B2, TyrA4B4, TyrA4B6, TyrA6B4, TyrA6B6, TyrA6B8, TyrA8B6, andTyrA8B8. A set of 18 building blocks will be combined to provide 18spots of homogeneous immobilized building block, 153 spots of candidateartificial receptors with two building blocks, 816 spots of candidateartificial receptors with three building blocks, and 3,060 spots ofcandidate artificial receptors with four building blocks. The largenumbers of spots from sets of 10 and 18 building blocks are sufficientto provide a thorough test of microspotting to form candidate artificialreceptors and control spots.

[0445] It should be noted that, as used in this specification and theappended claims, the singular forms “a,” “an,” and “the” include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to a composition containing “a compound” includes amixture of two or more compounds. It should also be noted that the term“or” is generally employed in its sense including “and/or” unless thecontent clearly dictates otherwise.

[0446] It should also be noted that, as used in this specification andthe appended claims, the phrase “adapted and configured” describes asystem, apparatus, or other structure that is constructed or configuredto perform a particular task or adopt a particular configuration to. Thephrase “adapted and configured” can be used interchangeably with othersimilar phrases such as arranged and configured, constructed andarranged, adapted, constructed, manufactured and arranged, and the like.

[0447] All publications and patent applications in this specificationare indicative of the level of ordinary skill in the art to which thisinvention pertains.

[0448] The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

What is claimed is:
 1. A method of making a heterogeneous building blockarray, the method comprising: forming a plurality of spots on a solidsupport, the spots comprising a plurality of building blocks; coupling aplurality of building blocks to the solid support in the spots.
 2. Themethod of claim 1, further comprising mixing a plurality of activatedbuilding blocks and employing the mixture in forming the plurality ofspots.
 3. The method of claim 1, comprising applying individualactivated building blocks on the support.
 4. The method of claim 1,wherein forming comprises piezoelectric spotting, pin spotting, orelectromagnetic spotting.
 5. The method of claim 1, wherein the solidsupport comprises a glass plate or microscope slide.
 6. A method ofmaking a receptor surface, the method comprising: forming a region on asolid support, the region comprising a plurality of building blocks;coupling the plurality of building blocks to the solid support in theregion.
 7. The method of claim 6, further comprising mixing a pluralityof activated building blocks and employing the mixture in forming thereceptor surface.
 8. The method of claim 6, comprising applyingindividual activated building blocks to the support.
 9. The method ofclaim 6, wherein the solid support comprises a tube, plate, or well. 10.A method of making an artificial receptor, the method comprising:forming a region on a support, the region comprising a plurality ofbuilding blocks; coupling the plurality of building blocks to thesupport in the region.
 11. The method of claim 10, wherein the region isa spot.
 12. The method of claim 10, wherein the support comprises ascaffold and the region comprises a plurality of functional groups onthe scaffold.
 13. A method of using an artificial receptor comprising:contacting a first heterogeneous molecular array with a test ligand; thearray comprising: a support; and a plurality of spots of building blocksattached to the support; the spots of building blocks comprising aplurality of building blocks; and the building blocks being coupled tothe support; detecting binding of a test ligand to one or more spots;and selecting one or more of the binding spots as the artificialreceptor; wherein the building blocks in the array define a first set ofbuilding blocks, and the plurality of building blocks in the one or morebinding spots defines one or more selected binding combination ofbuilding blocks.
 14. The method of claim 13, wherein the artificialreceptor comprises a lead artificial receptor.
 15. The method of claim13, further comprising: determining the combinations of building blocksin the one or more binding spots; developing, based on the combinationsdetermined, one or more developed combinations of building blocksdistinct from those in the one or more selected combinations of buildingblocks; contacting a second heterogeneous molecular array with the testligand, the second heterogeneous molecular array comprising a pluralityof spots, the spots comprising a developed combination of buildingblocks; detecting binding of a test ligand to one or more spots of thesecond heterogeneous molecular array; and selecting one or more of thespots of the second heterogeneous molecular array as the artificialreceptor; wherein the building blocks in the second heterogeneousmolecular array define a second set of building blocks.
 16. The methodof claim 15, wherein the artificial receptor comprises a lead artificialreceptor.
 17. The method of claim 16, further comprising varying thestructure of the lead artificial receptor to increase binding speed orbinding affinity of the test ligand.
 18. The method of claim 14, furthercomprising varying the structure of the lead artificial receptor toincrease binding speed or binding affinity of the test ligand.
 19. Themethod of claim 13, wherein the first set of building blocks comprises asubset of a larger set of building blocks.
 20. The method of claim 15,wherein the first set of building blocks comprises a subset of a largerset of building blocks, the second subset of building blocks defines asubset of the larger set of building blocks, and the first subset is notequivalent to the second subset.
 21. The method of claim 13, wherein thespots comprise 2, 3, or 4 building blocks.
 22. The method of claim 15,wherein the spots of the second heterogeneous molecular array comprise3, 4, or 5 building blocks, and the spots of the second heterogeneousmolecular array comprise more building blocks than the binding spots.23. The method of claim 13, further comprising: identifying theplurality of building blocks making up the artificial receptor; couplingthe identified plurality of building blocks to a scaffold molecule;evaluating the scaffold artificial receptor for binding of the testligand.
 24. The method of claim 23, wherein: coupling comprises making aplurality of positional isomers of the building blocks on the scaffold;evaluating comprises comparing the plurality of the scaffold positionalisomer artificial receptors; and selecting one or more of the scaffoldpositional isomer artificial receptors as lead or working artificialreceptor.
 25. The method of claim 15, further comprising: identifyingthe plurality of building blocks making up the artificial receptor;coupling the identified plurality of building blocks to a scaffoldmolecule; evaluating the scaffold artificial receptor for binding of thetest ligand.
 26. The method of claim 25, wherein: coupling comprisesmaking a plurality of positional isomers of the building blocks on thescaffold; evaluating comprises comparing the plurality of the scaffoldpositional isomer artificial receptors; and selecting one or more of thescaffold positional isomer artificial receptors as lead or workingartificial receptor.
 27. The method of claim 13, further comprisingapplying the test ligand to one or more spots that function as controlsfor validating or evaluating binding to an artificial receptor.
 28. Themethod of claim 27, wherein the control spot comprises no buildingblock, only a single building block, only functionalized lawn, or acombination thereof.
 29. A composition comprising: a support; and aportion of the support comprising a plurality of building blocks; thebuilding blocks being coupled to the support.
 30. The composition ofclaim 29, comprising a candidate artificial receptor, a lead artificialreceptor, a working artificial receptor, or a combination thereof. 31.The composition of claim 30, wherein the support comprises a scaffoldmolecule.
 32. The composition of claim 30, wherein the artificialreceptor comprises 2, 3, 4, 5, or 6 different building blocks.
 33. Thecomposition of claim 29, comprising a plurality of spots on the support;the spots comprising a plurality of building blocks; and the buildingblocks being coupled to the support.
 34. The composition of claim 33,wherein the spots are configured in an array.
 35. The composition ofclaim 34, wherein the array comprises more than 1 million spots.
 36. Thecomposition of claim 33, wherein the spots comprise 2, 3, 4, 5, or 6building blocks.
 37. The composition of claim 33, wherein the supportcomprises a solid support.
 38. The composition of claim 37, comprising aplurality of spots on a surface of the solid support.
 39. Thecomposition of claim 33, comprising a functionalized lawn coupled to thesupport and the building blocks coupled in spots to the lawn.
 40. Thecomposition of claim 39, comprising a functionalized glass support. 41.The composition of claim 33, wherein the support comprises a scaffoldmolecule.
 42. The composition of claim 29, wherein: the supportcomprises a surface; the surface comprises a region; and the regioncomprises a plurality of building blocks; the building blocks beingcoupled to the support.
 43. The composition of claim 42, wherein theregion comprises 2, 3, 4, 5, or 6 building blocks.
 44. The compositionof claim 42, wherein the support comprises a tube or well.
 45. Thecomposition of claim 42, further comprising a functionalized lawncoupled to the tube or well and the building blocks coupled to the lawn.46. The composition of claim 29, the plurality of building blocksindependently comprising framework, linker, first recognition element,and second recognition element.
 47. The composition of claim 46, whereinthe framework comprises an amino acid.
 48. The composition of claim 47,wherein the amino acid comprises serine, threonine, or tyrosine.
 49. Thecomposition of claim 47, wherein the amino acid comprises tyrosine. 50.The composition of claim 46, wherein the linker has the formula(CH₂)_(n)C(O)—, with n=1-16.
 51. The composition of claim 46, whereinthe first recognition element and second recognition elementindependently are of formulas B1, B2, B3, B4, B5, B6, B7, B8, B9, A1,A2, A3, A4, A5, A6, A7, A8, or A9.
 52. The composition of claim 47,wherein the support comprises a support matrix and the support matrixcomprises a lawn of amines.
 53. The composition of claim 29, theplurality of building blocks independently having the formula:

in which: X is absent or C═O; Y is absent, NH, or O; Z is O; R₂ is H orCH₃; R₃ is CH₂ or CH₂-phenyl; RE₁ is B1, B2, B3, B4, B5, B6, B7, B8, B9,A1, A2, A3, A4, A5, A6, A7, A8, or A9; RE₂ is A1, A2, A3, A4, A5, A6,A7, A8, A9, B1, B2, B3, B4, B5, B6, B7, B8, or B9; and L is(CH₂)_(n)COOH, with n=1-16.
 54. The composition of claim 53 the buildingblocks being independently:4-{4-[(acetylamino-ethylcarbamoyl-methyl)-amino]-phenoxy}-butyric acid;4-(4-{[(3-cyclopentyl-propionylamino)-ethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-[4-({[2-(3-chloro-phenyl)-acetylamino]-ethylcarbamoyl-methyl}-amino)-phenoxy]-butyricacid;4-(4-{[ethylcarbamoyl-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[ethylcarbamoyl-(3-pyridin-3-yl-propionylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[ethylcarbamoyl-(2-methylsulfanyl-acetylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[ethylcarbamoyl-(3-hydroxy-butyrylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-carbamoyl-propionylamino)-ethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(4-dimethylamino-butyrylamino)-ethylcarbarnoyl-methyl]-amino}-phenoxy)-butyricacid;4-{4-[(acetylamino-isobutylcarbamoyl-methyl)-amino]-phenoxy}-butyricacid;4-(4-{[(3-cyclopentyl-propionylamino)-isobutylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-[4-({[2-(3-chloro-phenyl)-acetylamino]-isobutylcarbamoyl-methyl}-amino)-phenoxy]-butyricacid;4-(4-{[isobutylcarbamoyl-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-1{[isobutylcarbamoyl-(3-pyridin-3-yl-propionylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[isobutylcarbamoyl-(2-methylsulfanyl-acetylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-hydroxy-butyrylamino)-isobutylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-(3-{[(3-carbamoyl-propionylamino)-isobutylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(4-dimethylamino-butyrylamino)-isobutylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-{4-[(acetylamino-phenethylcarbamoyl-methyl)-amino]-phenoxy}-butyricacid;4-(4-{[(3-cyclopentyl-propionylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-[4-({[2-(3-chloro-phenyl)-acetylamino]-phenethylcarbamoyl-methyl}-amino)-phenoxy]butyricacid;4-(4-{[phenethylcarbamoyl-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[phenethylcarbamoyl-(3-pyridin-3-yl-propionylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(2-methylsulfanyl-acetylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-hydroxy-butyrylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-carbamoyl-propionylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(4-dimethylamino-butyrylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)butyricacid;4-[4-({acetylamino-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}-amino)-phenoxy]butyricacid;4-[4-({(3-cyclopentyl-propionylamino)-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}amino)-phenoxy]-butyricacid;4-[4-({[2-(3-chloro-phenyl)-acetylamino]-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}amino)-phenoxy]-butyricacid;4-(4-{[[2-(4-methoxy-phenyl)-ethylcarbamoyl]-(3-phenyl-acryloylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[[2-(4-methoxy-phenyl)-ethylcarbamoyl]-(3-pyridin-3-yl-propionylamino)-methyl]amino}-phenoxy)-butyricacid;4-(4-{[[2-(4-methoxy-phenyl)-ethylcarbamoyl]-(2-methylsulfanyl-acetylamino)-methyl]amino}-phenoxy)-butyricacid;4-[4-({(3-hydroxy-butyrylamino)-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}-amino)phenoxy]-butyricacid;4-[4-({(3-carbamoyl-propionylamino)-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}amino)-phenoxy]-butyricacid;4-[4-({(4-dimethylamino-butyrylamino)-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}-amino)-phenoxy]-butyricacid;4-(4-{[acetylamino-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-cyclopentyl-propionylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid; 4-(4-{[[2-(3-chloro-phenyl)-acety lamino](2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyric acid;4-(4-{[(3-phenyl-acryloylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(2-pyridin-2-yl-ethylcarbamoyl)-(3-pyridin-3-yl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-methylsulfanyl-acetylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-hydroxy-butyrylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(3-carbamoyl-propionylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(4-dimethylamino-butyrylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[acetylamino-(2-methoxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-cyclopentyl-propionylamino)-(2-methoxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[[2-(3-chloro-phenyl)-acetylamino]-(2-methoxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-methoxy-ethylcarbamoyl)-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(2-methoxy-ethylcarbamoyl)-(3-pyridin-3-yl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-methoxy-ethylcarbamoyl)-(2-methylsulfanyl-acetylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(3-hydroxy-butyrylamino)-(2-methoxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;4-(3-{[(3-carbamoyl-propionylamino)-(2-methoxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(4-dimethylamino-butyrylamino)-(2-methoxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[acetylamino-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-cyclopentyl-propionylamino)-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[[2-(3-chloro-phenyl)-acetylamino]-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-hydroxy-ethylcarbamoyl)-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(2-hydroxy-ethylcarbamoyl)-(3-pyridin-3-yl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-hydroxy-ethylcarbamoyl)-(2-methylsulfanyl-acetylamino)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(3-hydroxy-butyrylamino)-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;4-(3-{[(3-carbamoyl-propionylamino)-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(4-dimethylamino-butyrylamino)-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[acetylamino-(2-acetylamino-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(2-acetylamino-ethylcarbamoyl)-(3-cyclopentyl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;4-[4-({(2-acetylamino-ethylcarbamoyl)-[2-(3-chloro-phenyl)-acetylamino]-methyl}-amino)phenoxy]-butyricacid;4-(4-{[(2-acetylamino-ethylcarbamoyl)-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(2-acetylamino-ethylcarbamoyl)-(3-pyridin-3-yl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-acetylamino-ethylcarbamoyl)-(2-methylsulfanyl-acetylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(2-acetylamino-ethylcarbamoyl)-(3-hydroxy-butyrylamino)-methyl]-amino}-phenoxy)butyricacid;4-(3-{[(2-acetylamino-ethylcarbamoyl)-(3-carbamoyl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-acetylamino-ethylcarbamoyl)-(4-dimethylamino-butyrylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[acetylamino-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-cyclopentyl-propionylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[[2-(3-chloro-phenyl)-acetylamino]-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(3-phenyl-acryloylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(3-pyridin-3-yl-propionylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(2-methylsulfanyl-acetylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(3-hydroxy-butyrylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(3-{[(3-carbamoyl-propionylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(4-dimethylamino-butyrylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid; salt thereof, ester thereof, or protected derivative thereof. 55.The composition of claim 29, wherein the support comprises a scaffoldmolecule.
 56. The composition of claim 29, wherein the artificialreceptor comprises 2, 3, 4, 5, or 6 different building blocks.
 57. Thecomposition of claim 29, wherein the support comprises a solid support.58. The composition of claim 29, comprising a functionalized lawncoupled to the support and the building blocks coupled in spots to thelawn.
 59. The composition of claim 58, comprising a functionalized glasssupport.
 60. An artificial receptor, the artificial receptor comprisinga plurality of building blocks coupled to a support.
 61. A heterogeneousbuilding block array comprising: a support; and a plurality of spots onthe support; the spots comprising a plurality of building blocks; andthe building blocks being coupled to the support.
 62. A compositioncomprising: a surface; and a region on the surface comprising aplurality of building blocks; the building blocks being coupled to thesupport.
 63. A composition of matter comprising a plurality of buildingblocks; the building blocks having the formula: linker-framework-(firstrecognition element)(second recognition element).
 64. The composition ofmatter of claim 63, wherein the framework comprises an amino acid. 65.The composition of matter of claim 64, wherein the amino acid comprisesserine, threonine, or tyrosine.
 66. The composition of matter of claim64, wherein the amino acid comprises tyrosine.
 67. The composition ofmatter of claim 63, wherein the linker has the formula (CH₂)_(n)CO—,with n=1-16.
 68. The composition of matter of claim 63, wherein thefirst recognition element and second recognition element independentlyare of formulas B1, B2, B3, B4, B5, B6, B7, B8, B9, A1, A2, A3, A4, A5,A6, A7, A8, or A9.
 69. The composition of matter of claim 63, theplurality of building blocks independently having the formula:

in which: X is absent or C═O; Y is absent, NH, or O; Z is O; R₂ is H orCH₃; R₃ is CH₂ or CH₂-phenyl; RE₁ is B1, B2, B3, B4, B5, B6, B7, B8, B9,A1, A2, A3, A4, A5, A6, A7, A8, or A9; RE₂ is A1, A2, A3, A4, A5, A6,A7, A8, A9, B1, B2, B3, B4, B5, B6, B7, B8, or B9; and L is(CH₂)_(n)COOH, with n=1-16.
 70. The composition of matter of claim 63,the building blocks being independently:4-{4-[(acetylamino-ethylcarbamoyl-methyl)-amino]-phenoxy}-butyric acid;4-(4-{[(3-cyclopentyl-propionylamino)-ethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-[4-({[2-(3-chloro-phenyl)-acetylamino]-ethylcarbamoyl-methyl}-amino)-phenoxy]-butyricacid;4-(4-{[ethylcarbamoyl-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[ethylcarbamoyl-(3-pyridin-3-yl-propionylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[ethylcarbamoyl-(2-methylsulfanyl-acetylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[ethylcarbamoyl-(3-hydroxy-butyrylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-carbamoyl-propionylamino)-ethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(4-dimethylamino-butyrylamino)-ethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-{4-[(acetylamino-isobutylcarbamoyl-methyl)-amino]-phenoxy}-butyricacid;4-(4-{[(3-cyclopentyl-propionylamino)-isobutylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-[4-({[2-(3-chloro-phenyl)-acetylamino]-isobutylcarbamoyl-methyl}-amino)-phenoxy]-butyricacid;4-(4-{[isobutylcarbamoyl-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[isobutylcarbamoyl-(3-pyridin-3-yl-propionylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[isobutylcarbamoyl-(2-methylsulfanyl-acetylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-hydroxy-butyrylamino)-isobutylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-(3-{[(3-carbamoyl-propionylamino)-isobutylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(4-dimethylamino-butyrylamino)-isobutylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-{4-[(acetylamino-phenethylcarbamoyl-methyl)-amino]-phenoxy}-butyricacid;4-(4-{[(3-cyclopentyl-propionylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-[4-({[2-(3-chloro-phenyl)-acetylamino]-phenethylcarbamoyl-methyl}-amino)-phenoxy]butyricacid;4-(4-{[phenethylcarbamoyl-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[phenethylcarbamoyl-(3-pyridin-3-yl-propionylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(2-methylsulfanyl-acetylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-hydroxy-butyrylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-carbamoyl-propionylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(4-dimethylamino-butyrylamino)-phenethylcarbamoyl-methyl]-amino}-phenoxy)butyricacid;4-[4-({acetylamino-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}-amino)-phenoxy]butyricacid;4-[4-({(3-cyclopentyl-propionylamino)-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}amino)-phenoxy]-butyricacid;4-[4-({[2-(3-chloro-phenyl)-acetylamino]-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}amino)-phenoxy]-butyricacid;4-(4-{[[2-(4-methoxy-phenyl)-ethylcarbamoyl]-(3-phenyl-acryloylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[[2-(4-methoxy-phenyl)-ethylcarbamoyl]-(3-pyridin-3-yl-propionylamino)-methyl]amino}-phenoxy)-butyricacid;4-(4-{[[2-(4-methoxy-phenyl)-ethylcarbamoyl]-(2-methylsulfanyl-acetylamino)-methyl]amino}-phenoxy)-butyricacid;4-[4-({(3-hydroxy-butyrylamino)-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}-amino)phenoxy]-butyricacid;4-[4-({(3-carbamoyl-propionylamino)-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}amino)-phenoxy]-butyricacid;4-[4-({(4-dimethylamino-butyrylamino)-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-methyl}amino)-phenoxy]-butyricacid;4-(4-{[acetylamino-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-cyclopentyl-propionylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[[2-(3-chloro-phenyl)-acetylamino]-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(3-phenyl-acryloylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(2-pyridin-2-yl-ethylcarbamoyl)-(3-pyridin-3-yl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-methylsulfanyl-acetylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-hydroxy-butyrylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(3-carbamoyl-propionylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(4-dimethylamino-butyrylamino)-(2-pyridin-2-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[acetylamino-(2-methoxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-cyclopentyl-propionylamino)-(2-methoxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[[2-(3-chloro-phenyl)-acetylamino]-(2-methoxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-methoxy-ethylcarbamoyl)-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(2-methoxy-ethylcarbamoyl)-(3-pyridin-3-yl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-methoxy-ethylcarbamoyl)-(2-methylsulfanyl-acetylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(3-hydroxy-butyrylamino)-(2-methoxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;4-(3-{[(3-carbamoyl-propionylamino)-(2-methoxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(4-dimethylamino-butyrylamino)-(2-methoxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[acetylamino-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-cyclopentyl-propionylamino)-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[[2-(3-chloro-phenyl)-acetylamino]-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-hydroxy-ethylcarbamoyl)-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(2-hydroxy-ethylcarbamoyl)-(3-pyridin-3-yl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-hydroxy-ethylcarbamoyl)-(2-methylsulfanyl-acetylamino)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(3-hydroxy-butyrylamino)-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;4-(3-{[(3-carbamoyl-propionylamino)-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(4-dimethylamino-butyrylamino)-(2-hydroxy-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[acetylamino-(2-acetylamino-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(2-acetylamino-ethylcarbamoyl)-(3-cyclopentyl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;4-[4-({(2-acetylamino-ethylcarbamoyl)-[2-(3-chloro-phenyl)-acetylamino]-methyl}-amino)phenoxy]-butyricacid;4-(4-{[(2-acetylamino-ethylcarbamoyl)-(3-phenyl-acryloylamino)-methyl]-amino}-phenoxy)butyricacid;4-(4-{[(2-acetylamino-ethylcarbamoyl)-(3-pyridin-3-yl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-acetylamino-ethylcarbamoyl)-(2-methylsulfanyl-acetylamino)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(2-acetylamino-ethylcarbamoyl)-(3-hydroxy-butyrylamino)-methyl]-amino}-phenoxy)butyricacid;4-(3-{[(2-acetylamino-ethylcarbamoyl)-(3-carbamoyl-propionylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-acetylamino-ethylcarbamoyl)-(4-dimethylamino-butyrylamino)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[acetylamino-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}-phenoxy)-butyricacid;4-(4-{[(3-cyclopentyl-propionylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[[2-(3-chloro-phenyl)-acetylamino]-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(3-phenyl-acryloylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(3-pyridin-3-yl-propionylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(2-methylsulfanyl-acetylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(3-hydroxy-butyrylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(3-{[(3-carbamoyl-propionylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid;4-(4-{[(4-dimethylamino-butyrylamino)-(2-pyrrolidin-1-yl-ethylcarbamoyl)-methyl]-amino}phenoxy)-butyricacid; salt thereof, ester thereof, or protected derivative thereof. 71.The composition of matter of claim 63, comprising about 10 to about 200distinct building blocks.
 72. The composition of matter of claim 63,wherein the building blocks are activated for coupling to a functionalgroup.
 73. The composition of matter of claim 63, wherein the buildingblocks are coupled to a support.
 74. The composition of matter of claim63, wherein each building block is in a container.
 75. The compositionof matter of claim 63, further comprising a package containing theplurality of building blocks and instructions for their use.
 76. Thecomposition of matter of claim 75, wherein the building blocks arecomponents of a heterogeneous molecular array.
 77. The composition ofmatter of claim 63, comprising a mixture of building blocks.